C. novyi for the treatment of solid tumors in humans

ABSTRACT

The present invention provides, inter alia, methods for treating or ameliorating an effect of a solid tumor present in a human. These methods include administering intratumorally to the human a unit dose of C. novyi, preferably C. novyi NT, colony forming units (CFUs), which contains about 1×103-1×107 CFUs suspended in a pharmaceutically acceptable carrier or solution. Methods for debulking a solid tumor present in a human, methods for ablating a solid tumor present in a human, a method for microscopically precise excision of tumor cells in a human, methods for treating or ameliorating an effect of a solid tumor that has metastasized to one or more sites in a human, unit doses of C. novyi, preferably C. novyi NT, CFUs, and kits for treating or ameliorating an effect of a solid tumor present in a human are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/781,273, filed Sep. 29, 2015, which is a U.S. National StageApplication of International Application No. PCT/US2014/032196, filedMar. 28, 2014, which claims benefit to U.S. Provisional Application No.61/806,497 filed Mar. 29, 2013. The entire contents of the aboveapplications are incorporated by reference as if recited in full herein.

FIELD OF INVENTION

The present invention provides, inter alia, methods for treating orameliorating an effect of a solid tumor present in a human, fordebulking a solid tumor present in a human, for microscopically preciseexcising of tumor cells in a human, and for ablating a solid tumorpresent in a human. Unit doses of C. novyi CFUs and kits are alsoprovided.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed as sequence listing text file“1065272-000591-seq.txt”, file size of 44 KB, created on Jun. 18, 2020.The aforementioned sequence listing is hereby incorporated by referencein its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

Strategies that successfully target and destroy human cancers recognizedifferences between normal and malignant tissues (Dang et al., 2001).Such differences can be found at the molecular level, as is the casewith genetic aberrations, or more holistically, as with thephysiological aberrations in a tumor.

It is known that malignant solid tumors are usually composed of anecrotic core and a viable rim. Therapeutic interventions to date havefocused on the well-vascularized outer shell of the tumor, but few havetargeted the inner hypoxic core (Jain et al., 2001). The inner core of atumor has unique characteristics that differentiate it from normaltissues. The core has a poor vascular supply and is therefore deficientin nutrients and oxygen. As a site of active cellular necrosis, the lackof a functional vascular supply limits the clearance of noxious cellbreakdown and results in a low pH. Such an environment is not suitablefor growth of most human cells but is a rich environment for the growthof certain anaerobic bacteria. More than sixty-years ago, this conceptled investigators to inject spores of Clostridium histolyticus intotumor-bearing animals (Parker et al., 1947). Remarkably, the bacteriagerminated only in the necrotic core of the tumor and liquefied thetumors. In the 1950s and 1960s, spores from Clostridium butyricum wereinjected into patients with a variety of very advanced solid tumormalignancies (Mose, 1967; Mose, 1972). Many patients had significantgermination and destruction of large portions of their tumors, but thevery poor health and advanced stage of these patients made theirclinical management difficult and the absence of complete clinicalresponses subdued further pursuit of this approach.

Successful treatment of solid tumors remains an unfulfilled medicalgoal. Accordingly, there is a need to find treatments for solid tumors.The present invention is directed to meeting this and other needs.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating an effect of a solid tumor present in a human. This methodcomprises administering intratumorally to the human a unit dose of C.novyi colony forming units (CFUs) comprising about 1×10³-1×10⁷ CFUssuspended in a pharmaceutically acceptable carrier or solution.

Another embodiment of the present invention is a method for debulking asolid tumor present in a human. This method comprises administeringintratumorally to the human a unit dose of C. novyi CFUs comprisingabout 1×10³-1×10⁷ CFUs suspended in a pharmaceutically acceptablecarrier or solution.

An additional embodiment of the present invention is a method fordebulking a solid tumor present in a human. This method comprisesadministering intratumorally to the human one to four cycles of a unitdose of C. novyi NT spores comprising about 1×10⁴ spores per cycle, eachunit dose of C. novyi NT being suspended in a pharmaceuticallyacceptable carrier or solution.

A further embodiment of the present invention is a method for treatingor ameliorating an effect of a solid tumor present in a human. Thismethod comprises administering intratumorally to the human one to fourcycles of a unit dose of C. novyi NT spores comprising about 1×10⁴spores per cycle, each unit dose of C. novyi NT spores being suspendedin a pharmaceutically acceptable carrier or solution.

Another embodiment of the present invention is method for ablating asolid tumor present in a human. This method comprises administeringintratumorally to the human a unit dose of C. novyi CFUs comprisingabout 1×10³-1×10⁷ CFUs suspended in a pharmaceutically acceptablecarrier or solution, wherein the tumor is ablated leaving a margin ofnormal tissue.

A further embodiment of the present invention is a unit dose of C. novyiCFUs. This unit dose comprises about 1×10³-1×10⁷ CFUs in apharmaceutically acceptable carrier or solution, which is effective fortreating or ameliorating an effect of a solid tumor present in a human.

An additional embodiment of the present invention is a kit for treatingor ameliorating an effect of a solid tumor present in a human. This kitcomprises a unit dose of C. novyi CFUs comprising about 1×10³-1×10⁷ CFUsin a pharmaceutically acceptable carrier or solution and instructionsfor use of the kit.

Another embodiment of the present invention is a method formicroscopically precise excision of tumor cells in a human. This methodcomprises administering intratumorally to the human a unit dose of C.novyi NT colony forming units (CFUs) comprising about 1×10³-1×10⁷ CFUssuspended in a pharmaceutically acceptable carrier or solution.

A further embodiment of the present invention is a method for treatingor ameliorating an effect of a solid tumor that has metastasized to oneor more sites in a human. This method comprises administeringintratumorally to the human a unit dose of C. novyi NT colony formingunits (CFUs) comprising at least about 1×10³-1×10⁷ CFUs suspended in apharmaceutically acceptable carrier or solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show various images of canine osteosarcomas on the rightdistal radius/ulna of test subjects “Sasha” (FIG. 1A) and “Sampson”(FIG. 1B) after radiation treatment and intravenous (IV) C. novyi NTinjection.

FIG. 2A shows Kaplan-Meier curves showing survival of F433 Fisher ratsafter orthotopic implantation of a syngeneic glioma cell line (F98).Outer line—C. novyi-NT spores injected into tumor 12-15 days after tumorimplantation. Inner line—control. FIG. 2B shows bioluminescence (Xenogenimaging system) in three representative F433 Fisher rats afterorthotopic implantation of F98 glioma cell line. Images acquired on day0 (pretreatment—day of C. novyi—NT spore injection), day 1 after ITinjection of C. novyi-NT spores, and day 2 after IT injection of C.novyi-NT spores. FIG. 2C shows luciferase activity (count in millions)on day 0 (pretreatment), day 1 after IT injection of C. novyi-NT spores,and day 2 after IT injection of C. novyi-NT spores.

FIGS. 3A-B and 4A-B show germinated C. novyi-NT bacteria withinmicroscopic brain tumor lesions. In these Figures, gram stain showedvegetative C. novyi-NT bacteria (white or black arrowheads) localized intumor (T) and stellate micro-invasion (S), but not in normal braintissue (Br). FIG. 3A is a 100× magnification showing the interface oftumor and normal brain.

FIG. 3B is a 400× magnification showing the interface of tumor andnormal brain. FIG. 4A is a 100× magnification showing the interface ofnormal brain, tumor, and stellate micro-invasion of neoplastic tissue.FIG. 4B is a 400× magnification showing C. novyi-NT germination in astellate micro-invasive lesion.

FIG. 5 is a table of summary data for samples sequenced.

FIG. 6 is a table of copy number alterations in canine sarcomas.

FIGS. 7A-F are photographic and CT images from dog 11-R01 showing apartial response to C. novyi-NT therapy. Images span pre-treatment today 70 after first IT dose of C. novyi-NT spores. FIG. 7A shows apre-treatment image of the peripheral nerve sheath tumor. FIG. 7B showsabscess formation on day 3 of the study, with extent confined to tumor.FIG. 7C shows medical debridement following spontaneous abscess ruptureand discharge of necrotic and purulent material, which allowed healingby second intention. FIG. 7D shows that the wound has healed completelyby day 70 of the study and 77.6% reduction in tumor longest diameter wasnoted.

FIG. 7E is a pre-treatment CT image, taken 4 days before firsttreatment, which shows extent of tumor (circle) at the intersection ofpinna and cranium.

FIG. 7F is a post-treatment CT image on day 10 of the study showingalmost complete de-bulking of tumor.

FIGS. 8A-D are photographic and CT images from dog 04-R03 showing acomplete response to C. novyi-NT therapy. Images span pre-treatment today 60 after first IT dose of C. novyi-NT spores. FIG. 8A shows apre-treatment image of the soft tissue sarcoma. FIG. 8B shows a tumorlocalized abscess formed on day 15 of the study, 1 day after a thirddose of C. novyi-NT spores. FIG. 8C shows that tumor de-bulking wascomplete by day 27 of the study and healthy granulation tissue hadformed. FIG. 8D shows that the wound had healed completely by day 60 ofthe study, and no residual tumor was noted (complete response). FIG. 8Eis a pre-treatment CT image, taken 5 days before first treatment,showing extent of tumor (circle) on antebrachium. FIG. 8F is apost-treatment CT image on day 62 of the study showing complete loss oftumor mass.

FIG. 9 shows the size of dog 11-R01's tumor from initial IT dosing of C.novyi NT spores to completion of the clinical course.

FIG. 10A shows photographic (upper panels) and CT images (lower panels)of a canine soft tissue sarcoma on test subject “Drake” (04-R01) afterIT dosing of C. novyi NT spores. Circled regions of the CT imagesindicate tumor location. FIG. 10B shows the size of Drake's tumor frominitial IT dosing of C. novyi NT, through three subsequent doses, tocompletion of the clinical course.

FIG. 11 shows the size of dog 04-R03's tumor from initial IT dosing ofC. novyi NT spores, through two subsequent cycles, to completion of theclinical course.

FIG. 12A shows tumor size in eight test subjects (11-R02, 04-R02,26-R01, 16-R02, 04-R05, 16-R03, 11-R04, and 04-R06) over the clinicalcourse in which four cycles of IT C. novyi NT spores were administered.FIG. 12B shows tumor size in three test subjects (04-R08, 01-R02, and10-R02) for which data from a complete clinical course was not availabledue to necessary amputation or data cutoff.

FIG. 13 shows an injection scheme for tumors treated in the IT studydisclosed in Examples 6 and 7.

FIGS. 14A-D show CT and MRI images from a human patient. FIG. 14A showsa post-treatment CT with contrast on day 3 demonstrating evidence ofintra- and extra-medullary air collection. FIG. 14B shows apre-treatment MRI (T1 with gadolinium contrast) of the right upperhumerus showing a contrast enhancing mass involving the soft tissue andpossibly adjacent bone. FIG. 14C shows a post-treatment MRI on day 4demonstrating diminished contrast enhancement in the tumor mass comparedto baseline. FIG. 14D shows a post-treatment MRI on day 29 showinghomogenous non-enhancing mass consistent with ongoing necrosis. Tumor ishighlighted with arrowheads.

FIGS. 15A-D show extensive tumor necrosis in the human patient treatedwith C. novyi-NT spores. FIGS. 15A and 15B show a pre-treatment tumorbiopsy showing viable tumor (leiomyosarcoma) cells, 40× (A) and 100× (B)magnification, respectively. FIGS. 15C and 15D show a post-treatmenttumor biopsy, 4 days after IT injection of C. novyi-NT spores, showingextensive necrosis of tumor cells, 40× (A) and 100× (B) magnification,respectively.

FIGS. 16A-D show various aspects of the IT injection procedure using athree-tined needle. FIG. 16A shows a photograph of the three-tinedneedle. FIGS. 16B and 16C show computed tomography (CT) images of thetarget injection area before and after insertion of the needle.

FIG. 16D shows a magnified image of the three tines of the needle. FIG.16E shows a CT image with overlaying measurements for determininginsertion points of the needle.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating an effect of a solid tumor present in a human. This methodcomprises administering intratumorally to the human a unit dose of C.novyi colony forming units (CFUs) comprising about 1×10³-1×10⁷ CFUssuspended in a pharmaceutically acceptable carrier or solution.

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean subjecting an individual subject(e.g., a human patient) to a protocol, regimen, process or remedy, inwhich it is desired to obtain a physiologic response or outcome in thatsubject, e.g., a patient. In particular, the methods and compositions ofthe present invention may be used to slow the development of diseasesymptoms or delay the onset of the disease or condition, or halt theprogression of disease development. However, because every treatedsubject may not respond to a particular treatment protocol, regimen,process or remedy, treating does not require that the desiredphysiologic response or outcome be achieved in each and every subject orsubject, e.g., patient, population. Accordingly, a given subject orsubject, e.g., patient, population may fail to respond or respondinadequately to treatment.

As used herein, the terms “ameliorate”, “ameliorating” and grammaticalvariations thereof mean to decrease the severity of the symptoms of adisease in a subject.

As used herein, a “solid tumor” means an abnormal mass of cell growth.Solid tumors may occur anywhere in the body. Solid tumors may becancerous (malignant) or noncancerous (benign). Examples of solid tumorsaccording to the present invention include adrenocortical carcinoma,anal tumor/cancer, bladder tumor/cancer, bone tumor/cancer (such asosteosarcoma), brain tumor, breast tumor/cancer, carcinoid tumor,carcinoma, cervical tumor/cancer, colon tumor/cancer, endometrialtumor/cancer, esophageal tumor/cancer, extrahepatic bile ducttumor/cancer, Ewing family of tumors, extracranial germ cell tumor, eyetumor/cancer, gallbladder tumor/cancer, gastric tumor/cancer, germ celltumor, gestational trophoblastic tumor, head and neck tumor/cancer,hypopharyngeal tumor/cancer, islet cell carcinoma, kidney tumor/cancer,laryngeal tumor/cancer, leiomyosarcoma, leukemia, lip and oral cavitytumor/cancer, liver tumor/cancer (such as hepatocellular carcinoma),lung tumor/cancer, lymphoma, malignant mesothelioma, Merkel cellcarcinoma, mycosis fungoides, myelodysplastic syndrome,myeloproliferative disorders, nasopharyngeal tumor/cancer,neuroblastoma, oral tumor/cancer, oropharyngeal tumor/cancer,osteosarcoma, ovarian epithelial tumor/cancer, ovarian germ cell tumor,pancreatic tumor/cancer, paranasal sinus and nasal cavity tumor/cancer,parathyroid tumor/cancer, penile tumor/cancer, pituitary tumor/cancer,plasma cell neoplasm, prostate tumor/cancer, rhabdomyosarcoma, rectaltumor/cancer, renal cell tumor/cancer, transitional cell tumor/cancer ofthe renal pelvis and ureter, salivary gland tumor/cancer, Sezarysyndrome, skin tumors (such as cutaneous t-cell lymphoma, Kaposi'ssarcoma, mast cell tumor, and melanoma), small intestine tumor/cancer,soft tissue sarcoma, stomach tumor/cancer, testicular tumor/cancer,thymoma, thyroid tumor/cancer, urethral tumor/cancer, uterinetumor/cancer, vaginal tumor/cancer, vulvar tumor/cancer, and Wilms'tumor. Preferably, the solid tumor is selected from the group consistingof soft tissue sarcoma, hepatocellular carcinoma, breast cancer,pancreatic cancer, and melanoma. More preferably, the solid tumor is aleiomyosarcoma, such as a retroperitoneal leiomyosarcoma.

As used herein, a “unit dose” means the amount of a medicationadministered to a subject, e.g., a human, in a single dose.

As used herein, “C. novyi” means a bacteria belonging to species ofClostridium novyi or a bacteria derived therefrom. Clostridium novyi,which may be obtained commercially from, e.g., the ATCC (#19402), is agram-positive anaerobic bacterium. A bacterium derived from Clostridiumnovyi may be made by, e.g., screening native Clostridium novyi forclones that possess specific characteristics. Preferred C. novyibacteria are those which are non-toxic or minimally toxic to a subjectsuch as a mammal, e.g., a human. For example, a preferred C. novyi, C.novyi NT, is a bacteria derived from native Clostridium novyi that haslost its single systemic toxin (α-toxin) gene by, e.g., a geneticengineering process or through a selection procedure. C. novyi NT may bemade, for example, using the procedure disclosed in Dang et al., 2001and U.S. Pat. No. 7,344,710. Thus, the present invention includes C.novyi as well as C. novyi NT bacteria.

Pharmacokinetic studies indicate that C. novyi NT spores, if injectedintravenously, are rapidly cleared from the circulation (greater than99% spores are cleared within 1 hour) and sequestered within thereticulo-endothelial system. Long-term distribution studies reveal thatthese spores are eventually eliminated from all tissues within one year.Delivered in spore form (dormant stage), C. novyi NT germinates(transitions from the spore to the vegetative state) when exposed to thehypoxic regions of tumors. Thus, the toxicities of C. novyi NT areexpected to be greater in tumor-bearing than in healthy patients.

Healthy mice and rabbits showed no apparent clinical signs (morbidity,mortality, or clinical appearance) of toxicity regardless of treatmentdose when injected with C. novyi NT intravenously. However, examinationof tissues at necropsy revealed both gross and microscopic inflammatorychanges that appeared to be treatment-dose dependent. These findings,primarily in the liver, spleen and adrenals, were noted at doses of5×10⁸ spores/kg or greater. Healthy animals receiving lower doses showedno gross or microscopic abnormalities at necropsy. In animals thatreceived high doses, resolution of inflammation was already evident onday 28 and all signs of inflammation were absent in all animals by oneyear following administration. To determine if C. novyi NT spores wouldgerminate in non-tumor hypoxic tissue, studies in elderly mice withatherosclerotic plaques and experimental myocardial infarctions weretreated with C. novyi NT. There was no evidence of spore localization orgermination within these vascular lesions. At the conclusion of thestudy, no clinical or pathologic abnormalities (other than thepre-existing cardiovascular lesions) were noted in these mice. Thesestudies demonstrated that C. novyi NT caused no apparent clinical andminimal pathological toxicity in healthy animals.

Intravenous (IV) injection of spores into immune-competent tumor-bearingmice leads to lysis of the tumor and an intense inflammatory response.In mice, one of three outcomes is typically observed: One subset(25-35%) of mice are cured (no tumor recurrence after one year ofobservation) and develop long-term immunity to the original tumor(Agrawal et al., 2004). Another subset (65-75%) experience completeclinical responses, but undergo a recurrence with re-growth of theoriginal tumor. Finally, the remaining subset (0 to 20%, depending onthe experiment) undergoes tumor destruction, but develop significantclinical toxicity 2-5 days after the initiation of therapy. Relativelysimple measures, such as hydration, are adequate to reduce thistoxicity, often entirely eliminating these signs. Studies in largeranimals (rabbits) show the same cure and recurrence rates with C. novyiNT therapy, but do not show the life-threatening clinical toxicityobserved in a subset of mice. Treatment-related death was observed intumor-bearing mice, but not in rabbits, treated with C. novyi NT spores(Diaz et al., 2005). In these studies toxicity was related to both sporedose and tumor size. In moribund mice, no specific clinical laboratoryor pathologic end-organ damage was noted and the only significantfinding was hepatosplenomegaly. Cured mice had rare remnant inflammatorychanges in the liver and spleen, but were otherwise no different thanuntreated animals. These studies show that toxicity in tumor-bearinganimals can be pronounced (death) in mice with large tumors, but wasminimal in larger animals (rabbits), and was manageable in mice withhydration or antibiotics.

Previous work using C. novyi NT spores injected intravenously (1×10⁹spores/m²) as a single agent in tumor bearing dogs produced no lifethreatening toxicities. The dogs were maintained on fluid therapy (2-4ml/kg/hr) for several days post treatment which may have decreased thetoxicity. Unfortunately, there were no measurable tumor responses to thetreatment.

As used herein, “colony forming units” (“CFUs”) mean viable forms of thebacteria which will give rise to an aggregate of cells (or colonies).Such viable forms include vegetative and spore forms, and the presentinvention includes both forms used separately and in combination. Colonyforming unit assays are known in the art. See, e.g., Breed et al., 1916.Media for supporting the growth of C. novyi are commercially available,such as Reinforced Clostridial Medium (RCM) from Difco (BD, FranklinLakes, N.J.). As set forth above, the unit dose comprises from about1×10³-1×10⁷, such as about 1×10³-1×10⁴, about 1×10⁴-1×10⁵, about1×10⁵-1×10⁶, or about 1×10⁶-1×10⁷ , C. novyi CFUs.

In one aspect of this embodiment, the unit dose comprises from about1×10⁶-1×10⁷ C. novyi CFUs. In another aspect of this embodiment, theunit dose comprises about 1×10⁴ C. novyi CFUs. Surprisingly, the dosesdisclosed herein for human treatment are unexpectedly lower than wouldbe expected from simply extrapolating from our non-rodent models using1/6 of the non-rodent highest non-severely toxic does (HNSTD), as istypical for a starting dose therapeutic for oncology indications. See,e.g., Senderowicz, A. M., “Information needed to conduct first-in humanoncology trials in the United States: a view from a former FDA medicalreviewer.” Clin. Canc. Res., 2010, 16:1719-25.

Preferably, in the present invention the C. novyi is C. novyi NT.

In another aspect of this embodiment, the unit dose comprises about1×10⁶-1×10⁷ C. novyi NT spores. In a further aspect of this embodiment,the unit dose comprises about 1×10⁴ C. novyi NT spores.

In an additional aspect of this embodiment, the administering stepcomprises injecting the unit dose at a single location into the tumor.In another aspect of this embodiment, the administering step comprisesinjecting the unit dose at multiple unique locations, such as 2, 3, 4,5, 6, 7, 8, 9, 10 or more than 10 unique locations, into the tumor.Preferably, the administering step comprises injecting the unit dose at1-5 unique locations into the tumor, such as in the configurations shownin FIG. 13. In another preferred embodiment, the administering stepcomprises injecting the unit dose at 5 or more unique locations into thetumor. Multi-site injections may be carried out as disclosed herein,preferably with a multi-tined needle such as Quadra-Fuse® (Rex-Medical,Conshohocken, Pa.). In the present invention, the administering step, asnoted above, includes injections directly into the tumor, but othermethods for administering an active agent, such as C. novyi or C. novyiNT, to a tumor are also contemplated. Such methods include implantation,transdermal delivery, and transmucosal delivery.

In another aspect of this embodiment, the method further comprisesadministering a plurality of treatment cycles, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more than 30 cycles, tothe human, each treatment cycle comprising injecting one unit dose ofthe C. novyi CFUs, such as one unit dose of the C. novyi NT spores, intothe solid tumor. Preferably, 1-10 treatment cycles are administered.More preferably, 2-4 treatment cycles are administered. The intervalbetween each treatment cycle may be variable. In one preferredembodiment, the interval between each treatment cycle is about 5-100days. In another preferred embodiment, the interval between eachtreatment cycle is about 7 days.

In an additional aspect of this embodiment, the method further comprisesadministering intravenous (IV) fluids to the human before, during,and/or after each administration of the C. novyi CFUs, such as the C.novyi NT spores. IV fluids for hydrating the patients are disclosedherein and are well known in the art. Such fluids may be fluids that areisotonic with blood, such as, e.g., a 0.9% sodium chloride solution, orLactated Ringer's solution.

In another aspect of this embodiment, the method further comprisesproviding the human with a first course of antibiotics for a period oftime and at a dosage that is effective to treat or alleviate an adverseside effect caused by the C. novyi CFUs, such as the C. novyi NT spores.In the present invention an adverse side effect (or adverse event, whichis used interchangeably with adverse side effect) may include but is notlimited to infections (such as those caused by open wounds), vomiting,hematochezia, and fever.

In one preferred embodiment, the antibiotics are administered for twoweeks post C. novyi administration. Non-limiting examples of suchantibiotics include amoxicillin, clavulanate, metronidazole, andcombinations thereof.

In another preferred embodiment, the method further comprises providingthe human with a second course of antibiotics for a period of time andat a dosage that is effective to treat or alleviate an adverse sideeffect caused by the C. novyi. The second course of antibiotics may beinitiated after completion of the first course of antibiotics and iscarried out for 1-6 months, such as 3 months. Preferably, the antibioticused in the second course is doxycycline, but any antibiotic approved bya medical professional may be used.

In a further aspect of this embodiment, the method further comprises,using a co-treatment protocol by, e.g., administering to the human atherapy selected from the group consisting of chemotherapy, radiationtherapy, immunotherapy, and combinations thereof.

The C. novyi, e.g., the C. novyi NT spores, and the anti-cancer agent(s)used in the co-treatment therapy may be administered to the human,either simultaneously or at different times, as deemed most appropriateby a physician. If the C. novyi, e.g., the C. novyi NT spores, and theother anti-cancer agent(s) are administered at different times, forexample, by serial administration, then the C. novyi, e.g., the C. novyiNT spores, may be administered to the human before the other anti-canceragent. Alternatively, the other anti-cancer agent(s) may be administeredto the human before the C. novyi, e.g., the C. novyi NT spores.

As used herein, “chemotherapy” means any therapeutic regimen that iscompatible with the C. novyi, e.g., C. novyi NT, treatment of thepresent invention and that uses cytotoxic and/or cytostatic agentsagainst cancer cells or cells that are associated with or support cancercells. In a preferred embodiment, the chemotherapy comprisesadministering to the human an agent selected from the group consistingof an anti-metabolite, a microtubule inhibitor, a DNA damaging agent, anantibiotic, an anti-angiogenesis agent, a vascular disrupting agent, amolecularly targeted agent, and combinations thereof.

As used herein, an “anti-metabolite” is a substance that reduces orinhibits a cell's use of a chemical that is part of normal metabolism.Non-limiting examples of anti-metabolite agents or analogs thereofaccording to the present invention include antifolates, purineinhibitors, pyrimidine inhibitors, and combinations thereof.

As used herein, an “antifolate” is a substance that alters, reduces, orinhibits the use of folic acid (vitamin B₉) by cells. Non-limitingexamples of antifolates include methotrexate (DuraMed Pharmaceuticals,Inc.), pemetrexed (Eli Lilly), pralatrexate (Spectrum Pharmaceuticals),aminopterin (Sigma Aldrich), pharmaceutically acceptable salts thereof,and combinations thereof.

As used herein, a “purine” is a compound that contains a fusedsix-membered and a five-membered nitrogen-containing ring. Non-limitingexamples of purines that are important for cellular metabolism includeadenine, guanine, hypoxanthine, and xanthine. A “purine inhibitor” is asubstance that alters, reduces or suppresses the production of a purineor the use of a purine by a cell. Non-limiting examples of purineinhibitors include methotrexate (DuraMed Pharmaceuticals, Inc.),pemetrexed (Eli Lilly), hydroxyurea (Bristol-Myers Squibb),2-mercaptopurine (Sigma-Aldrich), 6-mercaptopurine (Sigma-Aldrich),fludarabine (Ben Venue Laboratories), clofarabine (Genzyme Corp.),nelarabine (GlaxoSmithKline), pralatrexate (Spectrum Pharmaceuticals),6-thioguanine (Gate Pharmaceuticals), forodesine (BioCrystPharmaceuticals), pentostatin (Bedford Laboratories), sapacitabine(Cyclacel Pharmaceuticals, Inc.), aminopterin (Sigma Aldrich),azathioprine (GlaxoSmithKline), pharmaceutically acceptable saltsthereof, and combinations thereof.

As used herein, a “pyrimidine” is a compound that contains asix-membered nitrogen-containing ring. Non-limiting examples ofpyrimidines that are important for cellular metabolism include uracil,thymine, cytosine, and orotic acid. A “pyrimidine inhibitor” is asubstance that alters, reduces, or suppresses the production of apyrimidine or the use of a pyrimidine by the a cell. Non-limitingexamples of pyrimidine inhibitors include 5-fluorouracil (TocrisBioscience), tegafur (LGM Pharma), capecitabine (Xeloda) (Roche),cladribine (LGM Pharma), gemcitabine (Eli Lilly), cytarabine (BedfordLaboratories), decitabine (Eisai Inc.), floxuridine (BedfordLaboratories), 5-azacytidine (Pharmion Pharmaceuticals), doxifluridine(Cayman Chemicals), thiarabine (Access Pharmaceuticals), troxacitabine(SGX Pharmaceuticals), raltitrexed (AstraZeneca), carmofur (Santa CruzBiotechnology, Inc.), 6-azauracil (MP Biomedicals, LLC),pharmaceutically acceptable salts thereof, and combinations thereof.

In a preferred aspect of the present invention, the anti-metaboliteagent is selected from the group consisting of 5-fluorouracil (TocrisBioscience), tegafur (LGM Pharma), capecitabine (Xeloda) (Roche),cladribine (LGM Pharma), methotrexate (DuraMed Pharmaceuticals, Inc.),pemetrexed (Eli Lilly), hydroxyurea (Bristol-Myers Squibb),2-mercaptopurine (Sigma-Aldrich), 6-mercaptopurine (Sigma-Aldrich),fludarabine (Ben Venue Laboratories), gemcitabine (Eli Lilly),clofarabine (Genzyme Corp.), cytarabine (Bedford Laboratories),decitabine (Eisai Inc.), floxuridine (Bedford Laboratories), nelarabine(GlaxoSmithKline), pralatrexate (Spectrum Pharmaceuticals),6-thioguanine (Gate Pharmaceuticals), 5-azacytidine (PharmionPharmaceuticals), doxifluridine (Cayman Chemicals), forodesine (BioCrystPharmaceuticals), pentostatin (Bedford Laboratories), sapacitabine(Cyclacel Pharmaceuticals, Inc.), thiarabine (Access Pharmaceuticals),troxacitabine (SGX Pharmaceuticals), raltitrexed (AstraZeneca),aminopterin (Sigma Aldrich), carmofur (Santa Cruz Biotechnology, Inc.),azathioprine (GlaxoSmithKline), 6-azauracil (MP Biomedicals, LLC),pharmaceutically acceptable salts thereof, and combinations thereof.

As used herein, a “microtubule inhibitor” is a substance that disruptsthe functioning of a microtubule, such as the polymerization or thedepolymerization of individual microtubule units. In one aspect of thepresent invention, the microtubule inhibitor may be selected from thegroup consisting of a microtubule-destabilizing agent, amicrotubule-stabilizing agent, and combinations thereof. A microtubuleinhibitor of the present invention may also be selected from the groupconsisting of a taxane, a vinca alkaloid, an epothilone, andcombinations thereof. Non-limiting examples of microtubule inhibitorsaccording to the present invention include BT-062 (Biotest), HMN-214 (D.Western Therapeutics), eribulin mesylate (Eisai), vindesine (Eli Lilly),EC-1069 (Endocyte), EC-1456 (Endocyte), EC-531 (Endocyte), vintafolide(Endocyte), 2-methoxyestradiol (EntreMed), GTx-230 (GTx), trastuzumabemtansine (Hoffmann-La Roche), crolibulin (Immune Pharmaceuticals),D1302A-maytansinoid conjugates (ImmunoGen), IMGN-529 (ImmunoGen),lorvotuzumab mertansine (ImmunoGen), SAR-3419 (ImmunoGen), SAR-566658(ImmunoGen), IMP-03138 (Impact Therapeutics), topotecan/vincristinecombinations (LipoCure), BPH-8 (Molecular Discovery Systems),fosbretabulin tromethamine (OXiGENE), estramustine phosphate sodium(Pfizer), vincristine (Pierre Fabre), vinflunine (Pierre Fabre),vinorelbine (Pierre Fabre), RX-21101 (Rexahn), cabazitaxel (Sanofi),STA-9584 (Synta Pharmaceuticals), vinblastine, epothilone A, patupilone(Novartis), ixabepilone (Bristol-Myers Squibb), Epothilone D (KosanBiosciences), paclitaxel (Bristol-Myers Squibb), docetaxel(Sanofi-Aventis), HAI abraxane, DJ-927 (Daiichi Sankyo), discodermolide(CAS No: 127943-53-7), eleutherobin (CAS No.: 174545-76-7),pharmaceutically acceptable salts thereof, and combinations thereof.

DNA damaging agents of the present invention include, but are notlimited to, alkylating agents, platinum-based agents, intercalatingagents, and inhibitors of DNA replication.

As used herein, an “alkylating agent” is a substance that adds one ormore alkyl groups (C_(n)H_(m), where n and m are integers) to a nucleicacid. In the present invention, an alkylating agent is selected from thegroup consisting of nitrogen mustards, nitrosoureas, alkyl sulfonates,triazines, ethylenimines, and combinations thereof. Non-limitingexamples of nitrogen mustards include mechlorethamine (Lundbeck),chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.),bendamustine (Astellas), ifosfamide (Baxter International), melphalan(Ligand), melphalan flufenamide (Oncopeptides), and pharmaceuticallyacceptable salts thereof. Non-limiting examples of nitrosoureas includestreptozocin (Teva), carmustine (Eisai), lomustine (Sanofi), andpharmaceutically acceptable salts thereof. Non-limiting examples ofalkyl sulfonates include busulfan (Jazz Pharmaceuticals) andpharmaceutically acceptable salts thereof. Non-limiting examples oftriazines include dacarbazine (Bayer), temozolomide (Cancer ResearchTechnology), and pharmaceutically acceptable salts thereof. Non-limitingexamples of ethylenimines include thiotepa (Bedford Laboratories),altretamine (MGI Pharma), and pharmaceutically acceptable salts thereof.Other alkylating agents include ProLindac (Access), Ac-225 BC-8(Actinium Pharmaceuticals), ALF-2111 (Alfact Innovation), trofosfamide(Baxter International), MDX-1203 (Bristol-Myers Squibb),thioureidobutyronitrile (CellCeutix), mitobronitol (Chinoin), mitolactol(Chinoin), nimustine (Daiichi Sankyo), glufosfamide (EleisonPharmaceuticals), HuMax-TAC and PBD ADC combinations (Genmab), BP-C1(Meabco), treosulfan (Medac), nifurtimox (Metronomx), improsulfantosilate (Mitsubishi tanabe Pharma), ranimustine (Mitsubishi tanabePharma), ND-01 (NanoCarrier), HH-1 (Nordic Nanovector), 22P1G cells andifosfamide combinations (Nuvilex), estramustine phosphate (Pfizer),prednimustine (Pfizer), lurbinectedin (PharmaMar), trabectedin(PharmaMar), altreatamine (Sanofi), SGN-CD33A (Seattle Genetics),fotemustine (Servier), nedaplatin (Shionogi), heptaplatin (Sk Holdings),apaziquone (Spectrum Pharmaceuticals), SG-2000 (Spirogen), TLK-58747(Telik), laromustine (Vion Pharmaceuticals), procarbazine (AlkemLaboratories Ltd.), and pharmaceutically acceptable salts thereof.

As used herein, a “platinum-based agent” is an anti-cancer substancethat contains the metal platinum and analogs of such substances. Theplatinum may be in any oxidation state. Platinum-based agents of thepresent invention include, but are not limited to,1,2-diaminocyclohexane (DACH) derivatives, phenanthroimidazole Pt(II)complexes, platiunum IV compounds, bi- and tri-nuclear platinumcompounds, demethylcantharidin-integrated platinum complexes,platinum-conjugated compounds, cisplatin nanoparticles and polymermicelles, sterically hindered platinum complexes, oxaliplatin(Debiopharm), satraplatin (Johnson Matthey), BBR3464 (NovuspharmaS.p.A.), ZD0473 (Astra Zeneca), cisplatin (Nippon Kayaku), JM-11(Johnson Matthey), PAD (cis-dichlorobiscyclopentylamine platinum (II)),MBA ((trans-1,2-diaminocyclohexane) bisbromoacetato platinum (II)), PHM((1,2-Cyclohexanediamine) malonato platinum (II)), SHP((1,2-Cyclohexanediamine) sulphato platinum (II)), neo-PHM((trans-R,R-1,2-Cyclohexanediamine) malonato platinum (II)), neo-SHP((trans-R,R-1,2-Cyclohexanediamine)sulphato platinum (II)), JM-82(Johnson Matthey), PYP ((1,2-Cyclohexanediamine) bispyruvato platinum(II)), PHIC ((1,2-Cyclohexanediamine) isocitrato platinum (II)), TRK-710((trans-R,R-1,2-cyclohexanediamine)[3-Acetyl-5-methyl-2,4(3H,5H)-furandionato] platinum (II)), BOP((1,2-Cyclooctanediamine) bisbromoacetato platinum (II)), JM-40 (JohnsonMatthey), enloplatin (UnionPharma), zeniplatin (LGM Pharma), CI-973(Parke-Davis), lobaplatin (Zentaris AG/Hainan Tianwang InternationalPharmaceutical), cycloplatam (LGM Pharma), WA2114R(miboplatin/lobaplatin) (Chembest Research Laboratories, Ltd.),heptaplatin (SK12053R) (SK Chemicals), TNO-6 (spiroplatin) (HaihangIndustry Co., Ltd.), ormaplatin (tetraplatin) (LGM Pharma), JM-9(iproplatin) (Johnson Matthey), BBR3610 (Novuspharma S.p.A.), BBR3005(Novuspharma S.p.A.), BBR3571 (Novuspharma S.p.A.), BBR3537 (NovuspharmaS.p.A.), aroplatin (L-NDDP) (BOC Sciences), Pt-ACRAMTU ({[Pt(en)CI(ACRAMTU-S)](NO₃)₂(en=ethane-1,2-diamine,ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea)}),cisplatin-loaded liposomes (LiPlasomes), SPI-077 (Alza), lipoplatin(Regulon), lipoxal (Regulon), carboplatin (Johnson Matthey), nedaplatin(Shionogi Seiyaku), miriplatin hydrate (Dainippon Sumitomo Pharma),ormaplatin (LGM Pharma), enloplatin (Lederle Laboratories), C1973(Parke-Davis), PEGylated cisplatin, PEGylated carboplatin, PEGylatedoxaliplatin, transplatin (trans-diamminedichloroplatinum(II);mixedZ:trans-[PtCl₂{Z—HN═C(OMe)Me}(NH₃)]), CD-37 (estradiol-platinum(II)hybrid molecule), picoplatin (Poniard Pharmaceuticals),

AH44 (Komeda et al., 2006; Harris et al., 2005; Qu et al., 2004),triplatinNC (Harris et al., 2005; Qu et al., 2004), ProLindac (Access),pharmaceutically acceptable salts thereof, and combinations thereof.

As used herein, an “intercalating agent” includes, but is not limitedto, doxorubicin (Adriamycin), daunorubicin, idarubicin, mitoxantrone,pharmaceutically acceptable salts thereof, prodrugs, and combinationsthereof.

Non-limiting examples of inhibitors of DNA replication include, but arenot limited to topoisomerase inhibitors. As used herein, a“topoisomerase inhibitor” is a substance that decreases the expressionor the activity of a topoisomerase. The topoisomerase inhibitorsaccording to the present invention may inhibit topoisomerase I,topoisomerase II, or both topoisomerase I and topoisomerase II.Non-limiting examples of topoisomerase I inhibitors according to thepresent invention include irinotecan (Alchemia), APH-0804 (Aphios),camptothecin (Aphios), cositecan (BioNumerik), topotecan(GlaxoSmithKline), belotecan hydrochloride (Chon Kun Dang), firtecanpegol (Enzon), HN-30181A (Hanmi), hRS7-SN-38 (Immunomedics),labetuzumab-SN-38 (Immunomedics), etirinotecan pegol (NektarTherapeutics), NK-012 (Nippon Kayaku), SER-203 (Serina Therapeutics),simmitecan hydrochloride prodrug (Shanghai HaiHe Pharmaceuticals),gimatecan (Sigma-Tau), namitecan (Sigma-Tau), SN-38 (Supratek Pharma),TLC-388 hydrochloride (Taiwan Liposome Company), lamellarin D(PharmaMar), pharmaceutically acceptable salts thereof, and combinationsthereof. Non-limiting examples of inhibitors of topoisomerase type IIaccording to the present invention include Adva-27a (Advanomics),zoptarelin doxorubicin (Aeterna Zentaris), valrubicin (AnthraPharmaceuticals), razoxane (AstraZeneca), doxorubicin (AvenaTherapeutics), amsacrine (Bristol-Myers Squibb), etoposide phosphate(Bristol-Myers Squibb), etoposide (Novartis), dexrazoxane (CancerResearch Technology), cytarabine/daunorubicin combination (CelatorPharmaceuticals), CAP7.1 (CellAct Pharma), aldoxorubicin (CytRx),amrubicin hydrochloride (Dainippon Sumitomo Pharma), vosaroxin(Dainippon Sumitomo Pharma), daunorubicin (Gilead Sciences),milatuzumab/doxorubicin combination (Immunomedics), aclarubicin (KyowaHakko Kirin), mitoxantrone (Meda), pirarubicin (Meiji), epirubicin(Pfizer), teniposide (Novartis), F-14512 (Pierre Fabre), elliptiniumacetate (Sanofi), zorubicin (Sanofi), dexrazoxane (TopoTarget),sobuzoxane (Zenyaku Kogyo), idarubicin (Pfizer), HU-331 (CaymanChemical), aurintricarboxylic acid (Sigma Aldrich), pharmaceuticallyacceptable salts thereof, and combinations thereof.

Chemotherapeutic antibiotics according to the present invention include,but are not limited to, actinomycin, anthracyclines, valrubicin,epirubicin, bleomycin, plicamycin, mitomycin, pharmaceuticallyacceptable salts thereof, prodrugs, and combinations thereof.

As used herein, the term “anti-angiogenesis agent” means any compoundthat prevents or delays nascent blood vessel formation from existingvessels. In the present invention, examples of anti-angiogenesis agentsinclude, but are not limited to, pegaptanib, ranibizumab, bevacizumab(avastin), carboxyamidotriazole, TNP-470, CM101, IFN-α, IL-12, plateletfactor 4, suramin, SU5416, thrombospondin, VEGFR antagonists,angiostatic steroids and heparin, cartilage-derived angiogenesisinhibitory factor, matrix metalloproteinase inhibitors, angiostatin,endostatin, 2-methoxyestradiol, tecogalan, prolactin, α_(v)β₃inhibitors, linomide, VEGF-Trap, aminosterols, cortisone, tyrosinekinase inhibitors, anti-angiogenic siRNA, inhibitors of the complementsystem, vascular disrupting agents, and combinations thereof.Preferably, the anti-angiogenesis agent is bevacizumab.

VEGFR antagonists of the present invention include, but are not limitedto, pazopanib, regorafenib, lenvatinib, sorafenib, sunitinib, axitinib,vandetanib, cabozantinib, vatalanib, semaxanib, ZD6474, SU6668,AG-013736, AZD2171, AEE788, MF1/MC-18F1, DC101/IMC-1C11, ramucirumab,and motesanib. VEGFR antagonists may also include, VEGF inhibitors suchas bevacizumab, aflibercept, 2C3, r84, VEGF-Trap, and ranibizumab.

Angiostatic steroids of the present invention include any steroid thatinhibits, attenuates, prevents angiogenesis or neovascularization, orcauses regression of pathological vascularization. Angiostatic steroidsof the present invention include those disclosed in European PatentApplication Serial No. EP1236471 A2, as well as those 20-substitutedsteroids disclosed in U.S. Pat. No. 4,599,331, those 21-hydroxy steroidsdisclosed in U.S. Pat. No. 4,771,042, those C₁₁-functionalized steroidsdisclosed in International Application Serial No. WO 1987/02672,6α-fluoro17α,21-dihydroxy-16α-methylpregna-4,9(11)-diene-3,20-dione21-acetate,6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione,6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione21-phosphonooxy and pharmaceutically acceptable salts thereof,hydrocortisone, tetrahydrocortisol, 17α-hydroxy-progesterone,11α-epihydrocortisone, cortexolone, corticosterone,desoxycorticosterone, dexamethasone, cortisone 21-acetate,hydrocortisone 21-phosphate, 17α-hydroxy-6α-methylpregn-4-ene-3,20-dione17-acetate,6α-fluoro-17α,21-dihydroxy-16α-methylpregna-4,9(11)-diene-3,20-dione,and A9(11)-etianic esters, all disclosed in International ApplicationSerial No. WO 1990/015816 A1.

Cartilage-derived angiogenesis inhibitor factors include, but are notlimited to, peptide troponin and chondromodulin I.

Matrix metalloproteinase inhibitors of the present invention include,but are not limited to, succinyl hydroxamates such as marimastat andSC903, sulphonamide hydroxamates such as CGS27023A, phosphinamidehydroxamates, carboxylate inhibitors such as BAY12-9566, thiolinhibitors such as Compound B, aminomethyl benzimidazole analogues,peptides such as regasepin, and tetracyclines such as minocycline.

α_(v)β₃ inhibitors include, but are not limited to, IS201, P11 peptide,EMD 85189, and 66203, RGD peptide, RGD mimetics such as S 36578-2,echistatin, antibodies or antibody fragments against α_(v)β₃ integrinsuch as Vitaxin, which targets the extracellular domain of the dimer,cilengitide, and peptidomimetics such as S247.

Anti-angiogenic siRNAs include, but are not limited to, siRNAs targetingmRNAs that are upregulated during angiogenesis, optionally PEGylatedsiRNAs targeting VEGF or VEGFR mRNAs, and siRNAs targeting UPR (unfoldedprotein response)-IRE1α, XBP-1, and ATF6 mRNAs. Additionally, it hasbeen shown that siRNAs that are, at minimum, 21 nucleotides in length,regardless of targeting sequence, suppress neovascularization (Kleinman,et al., 2008) and may be included in the anti-angiogenic siRNAs of thepresent invention.

Inhibitors of the complement system include, but are not limited to,modified native complement components such as soluble complementreceptor type 1, soluble complement receptor type 1 lacking longhomologous repeat-A, soluble Complement Receptor Type 1-SialylLewis^(x), complement receptor type 2, soluble decay acceleratingfactor, soluble membrane cofactor protein, soluble CD59, decayaccelerating factor-CD59 hybrid, membrane cofactor protein-decayaccelerating factor hybrid, C1 inhibitor, and C1q receptor,complement-inhibitory antibodies such as anti-C5 monoclonal antibody andanti-C5 single chain Fv, synthetic inhibitors of complement activationsuch as antagonistic peptides and analogs targeting C5a receptor, andnaturally occurring compounds that block complement activation such asheparin and related glycosaminoglycan compounds. Additional inhibitorsof the complement system are disclosed by Makrides (Makrides, 1998).

As used herein, the term “vascular disrupting agent” means any compoundthat targets existing vasculature, e.g. tumor vasculature, damages ordestroys said vasculature, and/or causes central tumor necrosis. In thepresent invention, examples of vascular disrupting agents include, butare not limited to, ABT-751 (Abbott), AVE8062 (Aventis), BCN105(Bionomics), BMXAA (Antisoma), CA-4-P (OxiGene), CA-1-P (OxiGene),CYT997 (Cytopia), MPC-6827 (Myriad Pharmaceuticals), MN-029(MediciNova), NPI-2358 (Nereus), Oxi4503 (Oxigene), TZT-1027 (DaichiPharmaceuticals), ZD6126 (AstraZeneca and Angiogene), pharmaceuticallyacceptable salts thereof, and combinations thereof.

As used herein, a “molecularly targeted agent” is a substance thatinterferes with the function of a single molecule or group of molecules,preferably those that are involved in tumor growth and progression, whenadministered to a subject. Non-limiting examples of molecularly targetedagents of the present invention include signal transduction inhibitors,modulators of gene expression and other cellular functions, immunesystem modulators, antibody-drug conjugates (ADCs), and combinationsthereof.

As used herein, a “signal transduction inhibitor” is a substance thatdisrupts communication between cells, such as when an extracellularsignaling molecule activates a cell surface receptor. Non-limitingexamples of signal transduction inhibitors of the present inventioninclude anaplastic lymphoma kinase (ALK) inhibitors, B-Raf inhibitors,epidermal growth factor inhibitors (EGFRi), ERK inhibitors, Janus kinaseinhibitors, MEK inhibitors, mammalian target of rapamycin (mTor)inhibitors, phosphoinositide 3-kinase inhibitors (PI3Ki), and Rasinhibitors.

As used herein, an “anaplastic lymphoma kinase (ALK) inhibitor” is asubstance that (i) directly interacts with ALK, e.g., by binding to ALKand (ii) decreases the expression or the activity of ALK. Non-limitingexamples of anaplastic lymphoma kinase (ALK) inhibitors of the presentinvention include crizotinib (Pfizer, New York, N.Y.), CH5424802 (ChugaiPharmaceutical Co., Tokyo, Japan), GSK1838705 (GlaxoSmithKline, UnitedKingdom), Chugai 13d (Chugai Pharmaceutical Co., Tokyo, Japan), CEP28122(Teva Pharmaceutical Industries, Ltd., Israel), AP26113 (AriadPharmaceuticals, Cambridge, Mass.), Cephalon 30 (Teva PharmaceuticalIndustries, Ltd., Israel), X-396 (Xcovery, Inc., West Palm Beach, Fla.),Amgen 36 (Amgen Pharmaceuticals, Thousand Oaks, Calif.), ASP3026(Astellas Pharma US, Inc., Northbrook, Ill.), and Amgen 49 (AmgenPharmaceuticals, Thousand Oaks, Calif.), pharmaceutically acceptablesalts thereof, and combinations thereof.

As used herein, a “B-Raf inhibitor” of the present invention is asubstance that (i) directly interacts with B-Raf, e.g., by binding toB-Raf and (ii) decreases the expression or the activity of B-Raf. B-Rafinhibitors may be classified into two types by their respective bindingmodes. As used herein, “Type 1” B-Raf inhibitors are those inhibitorsthat target the ATP binding sites of the kinase in its activeconformation. “Type 2” B-Raf inhibitors are those inhibitors thatpreferentially bind to an inactive conformation of the kinase.Non-limiting examples of Type 1 B-Raf inhibitors of the presentinvention include:

(GlaxoSmithKline), GDC-0879 (Genentech), L-779450 B-Raf (Merck), PLX3202(Plexxikon), PLX4720 (Plexxikon), SB-590885 (GlaxoSmithKline), SB-699393(GlaxoSmithKline), vemurafenib (Plexxikon), pharmaceutically acceptablesalts thereof, and combinations thereof. Preferably, the type 1 RAFinhibitor is dabrafenib or a pharmaceutically acceptable salt thereof.

Non-limiting examples of Type 2 B-Raf inhibitors of the presentinvention include:

Sorafenib (Onyx Pharmaceuticals), ZM-336372 (AstraZeneca),pharmaceutically acceptable salts thereof, and combinations thereof

Other B-Raf inhibitors include, without limitation, AAL881 (Novartis);AB-024 (Ambit Biosciences), ARQ-736 (ArQule), ARQ-761 (ArQule), AZ628(Axon Medchem BV), BeiGene-283 (BeiGene), BIIB-024 (MLN 2480) (Sunesis &Takeda), b raf inhibitor (Sareum), BRAF kinase inhibitor (SelexagenTherapeutics), BRAF siRNA 313 (tacaccagcaagctagatgca) and 253(cctatcgttagagtcttcctg) (Liu et al., 2007), CTT239065 (Institute ofCancer Research), DP-4978 (Deciphera Pharmaceuticals), HM-95573 (Hanmi),GW 5074 (Sigma Aldrich), ISIS 5132 (Novartis), LErafAON (NeoPharm,Inc.), LBT613 (Novartis), LGX 818 (Novartis), pazopanib(GlaxoSmithKline), PLX5568 (Plexxikon), RAF-265 (Novartis), RAF-365(Novartis), regorafenib (Bayer Healthcare Pharmaceuticals, Inc.), RO5126766 (Hoffmann-La Roche), TAK 632 (Takeda), TL-241 (Teligene), XL-281(Exelixis), pharmaceutically acceptable salts thereof, and combinationsthereof.

As used herein, an “EGFR inhibitor” is a substance that (i) directlyinteracts with EGFR, e.g. by binding to EGFR and (ii) decreases theexpression or the activity of EGFR. Non-limiting examples of EGFRinhibitors according to the present invention include (+)-Aeroplysinin-1(CAS #28656-91-9), 3-(4-Isopropylbenzylidenyl)-indolin-2-one, ABT-806(Life Science Pharmaceuticals), AC-480 (Bristol-Myers Squibb), afatinib(Boehringer Ingelheim), AG 1478 (CAS #153436-53-4), AG 494 (CAS#133550-35-3), AG 555 (CAS #133550-34-2), AG 556 (CAS #133550-41-1), AG825 (CAS #149092-50-2), AG-490 (CAS #134036-52-5), antroquinonol (GoldenBiotechnology), AP-26113 (Ariad), ARRY334543 (CAS #845272-21-1), AST1306 (CAS #897383-62-9), AVL-301 (Celgene), AZD8931 (CAS #848942-61-0),BIBU 1361 (CAS #793726-84-8), BIBX 1382 (CAS #196612-93-8), BMS-690514(Bristol-Myers Squibb), BPIQ-I (CAS #174709-30-9), Canertinib (Pfizer),cetuximab (Actavis), cipatinib (Jiangsu Hengrui Medicine), CL-387,785(Santa Cruz Biotech), compound 56 (CAS #171745-13-4), CTX-023 (CytomXTherapeutics), CUDC-101 (Curis), dacomitinib (Pfizer), DAPH (CAS#145915-58-8), daphnetin (Santa Cruz Biotech), dovitinib lactate(Novartis), EGFR Inhibitor (CAS #879127-07-8), epitinib (Hutchison ChinaMediTech), erbstatin Analog (CAS #63177-57-1), erlotinib (Astellas),gefitinib (AstraZeneca), GT-MAB 5.2-GEX (Glycotope), GW 583340 (CAS#388082-81-3), GW2974 (CAS #202272-68-2), HDS 029 (CAS #881001-19-0),Hypericin (Santa Cruz Biotech), icotinib hydrochloride (Betapharma),JNJ-26483327 (Johnson & Johnson), JNJ-28871063 (Johnson & Johnson),KD-020 (Kadmon Pharmaceuticals), lapatinib ditosylate (GlaxoSmithKline),Lavendustin A (Sigma), Lavendustin C (Sigma), LY-3016859 (Eli Lilly),MEHD-7945A (Hoffmann-La Roche), MM-151 (Merrimack), MT-062 (MedisynTechnologies), necitumumab (Eli Lilly), neratinib (Pfizer), nimotuzumab(Center of Molecular Immunology), NT-004 (NewGen Therapeutics),pantiumumab (Amgen), PD 153035 (CAS #153436-54-5), PD 161570 (CAS#192705-80-9), PD 168393, PD 174265 (CAS #216163-53-0), pirotinib(Sihuan Pharmaceutical), poziotinib (Hanmi), PP 3 (CAS #5334-30-5),PR-610 (Proacta), pyrotinib (Jiangsu Hengrui Medicine), RG-13022 (CAS#136831-48-6), rindopepimut (Celldex Therapeutics), RPI-1 (CAS#269730-03-2), S-222611 (Shionogi), TAK 285 (CAS #871026-44-7), TAS-2913(Taiho), theliatinib (Hutchison China MediTech), Tyrphostin 47(RG-50864, AG-213) (CAS #118409-60-2), Tyrphostin 51 (CAS #122520-90-5),Tyrphostin AG 1478 (CAS #175178-82-2), Tyrphostin AG 183 (CAS#126433-07-6), Tyrphostin AG 528 (CAS #133550-49-9), Tyrphostin AG 99(CAS #118409-59-9), Tyrphostin B42 (Santa Cruz Biotech), Tyrphostin B44(Santa Cruz Biotech), Tyrphostin RG 14620 (CAS #136831-49-7), vandetanib(AstraZeneca), varlitinib (Array BioPharma), vatalanib (Novartis), WZ3146 (CAS #1214265-56-1), WZ 4002 (CAS #1213269-23-8), WZ8040 (CAS#1214265-57-2), XL-647 (Exelixis), Z-650 (HEC Pharm), ZM 323881 (CAS#324077-30-7), pharmaceutically acceptable salts thereof, andcombinations thereof. Preferably, the EGFR inhibitor is selected fromthe group consisting of panitumumab, erlotinib, pharmaceuticallyacceptable salts thereof, and combinations thereof.

As used herein, an “ERK inhibitor” is a substance that (i) directlyinteracts with ERK, including ERK1 and ERK2, e.g., by binding to ERK and(ii) decreases the expression or the activity of an ERK protein kinase.Therefore, inhibitors that act upstream of ERK, such as MEK inhibitorsand RAF inhibitors, are not ERK inhibitors according to the presentinvention. Non-limiting examples of ERK inhibitors of the presentinvention include AEZS-131 (Aeterna Zentaris), AEZS-136 (AeternaZentaris), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.),SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable saltsthereof, and combinations thereof.

As used herein, a “Janus kinase inhibitor” is a substance that (i)directly interacts with a Janus kinase, e.g., by binding to a Januskinase and (ii) decreases the expression or the activity of a Januskinase. Janus kinases of the present invention include Tyk2, Jak1, Jak2,and Jak3. Non-limiting examples of Janus kinase inhibitors of thepresent invention include ruxolitinib (Incyte Corporation, Wilmington,Del.), baricitinib (Incyte Corporation, Wilmington, Del.), tofacitinib(Pfizer, New York, N.Y.), VX-509 (Vertex Pharmaceuticals, Inc., Boston,Mass.), GLPG0634 (Galapagos NV, Belgium), CEP-33779 (TevaPharmaceuticals, Israel), pharmaceutically acceptable salts thereof, andcombinations thereof

As used herein, a “MEK inhibitor” is a substance that (i) directlyinteracts with MEK, e.g., by binding to MEK and (ii) decreases theexpression or the activity of MEK. Therefore, inhibitors that actupstream of MEK, such as RAS inhibitors and RAF inhibitors, are not MEKinhibitors according to the present invention. MEK inhibitors may beclassified into two types depending on whether the inhibitor competeswith ATP. As used herein, a “Type 1” MEK inhibitor is an inhibitor thatcompetes with ATP for binding to MEK. A “Type 2” MEK inhibitor is aninhibitor that does not compete with ATP for binding to MEK.Non-limiting examples of type 1 MEK inhibitors according to the presentinvention include bentamapimod (Merck KGaA), L783277 (Merck), R0092210(Roche), pharmaceutically acceptable salts thereof, and combinationsthereof. Preferably, the type 1 MEK inhibitor is RO092210 (Roche) or apharmaceutically acceptable salt thereof. Non-limiting examples of type2 MEK inhibitors according to the present invention include anthraxtoxin, lethal factor portion of anthrax toxin, ARRY-142886(6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide) (Array BioPharma), ARRY-438162 (ArrayBioPharma), AS-1940477 (Astellas), MEK162 (Array BioPharma), PD 098059(2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one), PD 184352(CI-1040), PD-0325901 (Pfizer), pimasertib (Santhera Pharmaceuticals),refametinib (AstraZeneca), selumetinib (AZD6244) (AstraZeneca), TAK-733(Takeda), trametinib (Japan Tobacco), U0126(1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene) (Sigma),RDEA119 (Ardea Biosciences/Bayer), pharmaceutically acceptable saltsthereof, and combinations thereof. Preferably, the type 2 MEK inhibitoris trametinib or a pharmaceutically acceptable salt thereof. Other MEKinhibitors include, without limitation, antroquinonol (GoldenBiotechnology), AS-1940477 (Astellas), AS-703988 (Merck KGaA), BI-847325(Boehringer Ingelheim), E-6201 (Eisai), GDC-0623 (Hoffmann-La Roche),GDC-0973, RG422, RO4987655, RO5126766, SL327, WX-554 (Wilex), YopJpolypeptide, pharmaceutically acceptable salts thereof, and combinationsthereof.

As used herein, an “mTOR inhibitor” is a substance that (i) directlyinteracts with mTOR, e.g. by binding to mTOR and (ii) decreases theexpression or the activity of mTOR. Non-limiting examples of mTORinhibitors according to the present invention include zotarolimus(AbbVie), umirolimus (Biosensors), temsirolimus (Pfizer), sirolimus(Pfizer), sirolimus NanoCrystal (Elan Pharmaceutical Technologies),sirolimus TransDerm (TransDerm), sirolimus-PNP (Samyang), everolimus(Novartis), biolimus A9 (Biosensors), ridaforolimus (Ariad), rapamycin,TCD-10023 (Terumo), DE-109 (MacuSight), MS-R001 (MacuSight), MS-R002(MacuSight), MS-R003 (MacuSight), Perceiva (MacuSight), XL-765(Exelixis), quinacrine (Cleveland BioLabs), PKI-587 (Pfizer),PF-04691502 (Pfizer), GDC-0980 (Genentech and Piramed), dactolisib(Novartis), CC-223 (Celgene), PWT-33597 (Pathway Therapeutics), P-7170(Piramal Life Sciences), LY-3023414 (Eli Lilly), INK-128 (Takeda),GDC-0084 (Genentech), DS-7423 (Daiichi Sankyo), DS-3078 (DaiichiSankyo), CC-115 (Celgene), CBLC-137 (Cleveland BioLabs), AZD-2014(AstraZeneca), X-480 (Xcovery), X-414 (Xcovery), EC-0371 (Endocyte),VS-5584 (Verastem), PQR-401 (Piqur), PQR-316 (Piqur), PQR-311 (Piqur),PQR-309 (Piqur), PF-06465603 (Pfizer), NV-128 (Novogen), nPT-MTOR(Biotica Technology), BC-210 (Biotica Technology), WAY-600 (BioticaTechnology), WYE-354 (Biotica Technology), WYE-687 (Biotica Technology),LOR-220 (Lorus Therapeutics), HMPL-518 (Hutchison China MediTech),GNE-317 (Genentech), EC-0565 (Endocyte), CC-214 (Celgene), and ABTL-0812(Ability Pharmaceuticals).

As used herein, a “PI3K inhibitor” is a substance that decreases theexpression or the activity of phosphatidylinositol-3 kinases (PI3Ks) ordownstream proteins, such as Akt. PI3Ks, when activated, phosphorylatethe inositol ring 3′-OH group in inositol phospholipids to generate thesecond messenger phosphatidylinositol-3,4,5-trisphosphate(PI-3,4,5-P(3)). Akt interacts with a phospholipid, causing it totranslocate to the inner membrane, where it is phosphorylated andactivated. Activated Akt modulates the function of numerous substratesinvolved in the regulation of cell survival, cell cycle progression andcellular growth.

Non-limiting examples of PI3K inhibitors according to the presentinvention include A-674563 (CAS #552325-73-2), AGL 2263, AMG-319 (Amgen,Thousand Oaks, Calif.), AS-041164(5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850(5-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione),AS-605240 (5-quinoxilin-6-methylene-1,3-thiazolidine-2,4-dione), AT7867(CAS #857531-00-1), benzimidazole series, Genentech (Roche HoldingsInc., South San Francisco, Calif.), BML-257 (CAS #32387-96-5), CAL-120(Gilead Sciences, Foster City, Calif.), CAL-129 (Gilead Sciences),CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (GileadSciences), CAS #612847-09-3, CAS #681281-88-9, CAS #75747-14-7, CAS#925681-41-0, CAS #98510-80-6, CCT128930 (CAS #885499-61-6), CH5132799(CAS #1007207-67-1), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK),FPA 124 (CAS #902779-59-3), GS-1101 (CAL-101) (Gilead Sciences), GSK690693 (CAS #937174-76-0), H-89 (CAS #127243-85-0), Honokiol, IC87114(Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (KarusTherapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1(Karus Therapeutics), KT 5720 (CAS #108068-98-0), Miltefosine, MK-2206dihydrochloride (CAS #1032350-13-2), ML-9 (CAS #105637-50-1),Naltrindole Hydrochloride, OXY-111A (NormOxys Inc., Brighton, Mass.),perifosine, PHT-427 (CAS #1191951-57-1), PI3 kinase delta inhibitor,Merck KGaA (Merck & Co., Whitehouse Station, N.J.), PI3 kinase deltainhibitors, Genentech (Roche Holdings Inc.), PI3 kinase deltainhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hydrabad, India),PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors,Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (RocheHoldings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics(Pathway Therapeutics Ltd., South San Francisco, Calif.), PI3-deltainhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-deltainhibitors, Intellikine (Intellikine Inc., La Jolla, Calif.), PI3-deltainhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.),PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway TherapeuticsLtd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG),PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gammainhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors,Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, PathwayTherapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors,Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitorEvotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gammainhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), pictilisib(GDC-0941) (Roche Holdings Inc.), PIK-90 (CAS #677338-12-4), SC-103980(Pfizer, New York, N.Y.), SF-1126 (Semafore Pharmaceuticals,Indianapolis, Ind.), SH-5, SH-6, Tetrahydro Curcumin, TG100-115(Targegen Inc., San Diego, Calif.), Triciribine, X-339 (Xcovery, WestPalm Beach, Fla.), XL-499 (Evotech, Hamburg, Germany), pharmaceuticallyacceptable salts thereof, and combinations thereof. Preferably, theinhibitor of the PI3K/Akt pathway is pictilisib (GDC-0941) or apharmaceutically acceptable salt thereof.

As used herein, a “RAS inhibitor” is a substance that (i) directlyinteracts with RAS, e.g., by binding to RAS and (ii) decreases theexpression or the activity of RAS. Non-limiting examples of RASinhibitors according to the present invention include farnesyltransferase inhibitors (such as, e.g., tipifarnib and lonafarnib),farnesyl group-containing small molecules (such as, e.g., salirasib andTLN-4601), DCAI, as described by Maurer (Maurer, et al., 2012), Kobe0065and Kobe2602, as described by Shima (Shima, et al., 2013), and HBS 3(Patgiri, et al., 2011), and AIK-4 (Allinky), pharmaceuticallyacceptable salts thereof, and combinations thereof.

As used herein, “gene expression” is a process by which the informationfrom DNA is used in the formation of a polypeptide. A “modulator of geneexpression and other cellular functions” is a substance that affectsgene expression and other works of a cell. Non-limiting examples of suchmodulators include hormones, histone deacetylase inhibitors (HDACi), andcyclin-dependent kinase inhibitors (CDKi), and poly ADP ribosepolymerase (PARP) inhibitors.

In the present invention, a “hormone” is a substance released by cellsin one part of a body that affects cells in another part of the body.Non-limiting examples of hormones according to the present inventioninclude prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin,antimullerian hormone, adiponectin, adrenocorticotropic hormone,angiotensinogen, angiotensin, vasopressin, atriopeptin, brainnatriuretic peptide, calcitonin, cholecystokinin,corticotropin-releasing hormone, encephalin, endothelin, erythropoietin,follicle-stimulating hormone, galanin, gastrin, ghrelin, glucagon,gonadotropin-releasing hormone, growth hormone-releasing hormone, humanchorionic gonadotropin, human placental lactogen, growth hormone,inhibin, insulin, somatomedin, leptin, liptropin, luteinizing hormone,melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreaticpolypeptide, parathyroid hormone, prolactin, prolactin releasinghormone, relaxin, renin, secretin, somatostain, thrombopoietin,thyroid-stimulating hormone, testosterone, dehydroepiandrosterone,androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone,estriol, cortisol, progesterone, calcitriol, and calcidiol.

Some compounds interfere with the activity of certain hormones or stopthe production of certain hormones. Non-limiting examples ofhormone-interfering compounds according to the present invention includetamoxifen (Nolvadex®), anastrozole (Arimidex®), letrozole (Femara®), andfulvestrant (Faslodex®). Such compounds are also within the meaning ofhormone in the present invention.

As used herein, an “HDAC inhibitor” is a substance that (i) directlyinteracts with HDAC, e.g., by binding to HDAC and (ii) decreases theexpression or the activity of HDAC. Non-limiting examples of HDACinhibitors according to the present invention include 4SC-201 (4SC AG),4SC-202 (Takeda), abexinostat (Celera), AN-1 (Titan Pharmaceuticals,Inc.), Apicidine (Merck & Co., Inc.), AR-42 (Arno Therapeutics),ARQ-700RP (ArQule), Avugane (TopoTarget AS),azelaic-1-hydroxamate-9-anilide (AAHA), belinostat (TopoTarget),butyrate (Enzo Life Sciences, Inc.), CG-1255 (Errant Gene Therapeutics,LLC), CG-1521 (Errant Gene Therapeutics, LLC), CG-200745(CrystalGenomics, Inc.), chidamide (Shenzhen Chipscreen Biosciences),CHR-3996 (Chroma Therapeutics), CRA-024781 (Pharmacyclics), CS-3158(Shenzhen Chipscreen Biosciences), CU-903 (Curis), DAC-60 (Genextra),entinostat (Bayer), hyaluronic acid butyric acid ester (HA-But), IKH-02(IkerChem), IKH-35 (IkerChem), ITF-2357 (Italfarmaco), ITF-A(Italfarmaco), JNJ-16241199 (Johnson & Johnson), KA-001 (KarusTherapeutics), KAR-3000 (Karus Therapeutics), KD-5150 (Kalypsys),KD-5170 (Kalypsys), KLYP-278 (Kalypsys), KLYP-298 (Kalypsys), KLYP-319(Kalypsys), KLYP-722 (Kalypsys), m-carboxycinnamic acid bis-hydroxamide(CBHA), MG-2856 (MethylGene), MG-3290 (MethylGene), MG-4230(MethylGene), MG-4915 (MethylGene), MG-5026 (MethylGene), MGCD-0103(MethylGene Inc.), mocetinostat (MethylGene), MS-27-275 (Schering AG),NBM-HD-1 (NatureWise), NVP-LAQ824 (Novartis), OCID-4681-S-01 (OrchidPharmaceuticals), oxamflatin ((2E)-5-[3-[(phenylsufonyl) aminolphenyl]-pent-2-en-4-ynohydroxamic acid), panobinostat (Novartis),PCI-34051 (Pharmacyclics), phenylbutyrate (Enzo Life Sciences, Inc.),pivaloyloxymethyl butyrate (AN-9, Titan Pharmaceuticals, Inc.), pivanex(Titan Pharmaceuticals, Inc.), pracinostat (SBIO), PX-117794 (TopoTargetAS), PXD-118490 (LEO-80140) (TopoTarget AS), pyroxamide(suberoyl-3-aminopyridineamide hydroxamic acid), resminostat (Takeda),RG-2833 (RepliGen), ricolinostat (Acetylon), romidepsin (Astellas),SB-1304 (S*BIO), SB-1354 (S*BIO), SB-623 (Merrion Research I Limited),SB-624 (Merrion Research I Limited), SB-639 (Merrion Research ILimited), SB-939 (S*BIO), Scriptaid(N-Hydroxy-1,3-dioxo-1H-benz[de]isoquinoline-2(3H)-hexan amide), SK-7041(In2Gen/SK Chemical Co.), SK-7068 (In2Gen/SK Chemical Co.),suberoylanilide hydroxamic acid (SAHA), sulfonamide hydroxamic acid,tributyrin (Sigma Aldrich), trichostatin A (TSA) (Sigma Aldrich),valporic acid (VPA) (Sigma Aldrich), vorinostat (Zolinza), WF-27082B(Fujisawa Pharmaceutical Company, Ltd.), pharmaceutically acceptablesalts thereof, and combinations thereof. Preferably, the HDAC inhibitoris romidepsin, pharmaceutically acceptable salts thereof, andcombinations thereof.

As used herein, “CDK” is a family of protein kinases that regulate thecell cycle. Known CDKs include cdk1, cdk2, ckd3, ckd4, cdk5, cdk6, cdk7,cdk8, cdk9, cdk10, and cdk11. A “CDK inhibitor” is a substance that (i)directly interacts with CDK, e.g. by binding to CDK and (ii) decreasesthe expression or the activity of CDK. Non-limiting examples of CDKinhibitors according to the present invention include 2-Hydroxybohemine,3-ATA, 5-Iodo-Indirubin-3′-monoxime, 9-Cyanopaullone, Aloisine A,Alsterpaullone 2-Cyanoethyl, alvocidib (Sanofi), AM-5992 (Amgen),Aminopurvalanol A, Arcyriaflavin A, AT-7519 (Astex Pharmaceuticals), AZD5438 (CAS #602306-29-6), BMS-265246 (CAS #582315-72-8), BS-181 (CAS#1092443-52-1), Butyrolactone I (CAS #87414-49-1), Cdk/Crk Inhibitor(CAS #784211-09-2), Cdk1/5 Inhibitor (CAS #40254-90-8), Cdk2 InhibitorII (CAS #222035-13-4), Cdk2 Inhibitor IV, NU6140 (CAS #444723-13-1),Cdk4 Inhibitor (CAS #546102-60-7), Cdk4 Inhibitor III (CAS#265312-55-8), Cdk4/6 Inhibitor IV (CAS #359886-84-3), Cdk9 Inhibitor II(CAS #140651-18-9), CGP 74514A, CR8, CYC-065 (Cyclacel), dinaciclib(Ligand), (R)-DRF053 dihydrochloride (CAS #1056016-06-8), Fascaplysin,Flavopiridol, Hygrolidin, Indirubin, LEE-011 (Astex Pharmaceuticals),LY-2835219 (Eli Lilly), milciclib maleate (Nerviano Medical Sciences),MM-D37K (Maxwell Biotech), N9-Isopropyl-olomoucine, NSC 625987 (CAS#141992-47-4), NU2058 (CAS #161058-83-9), NU6102 (CAS #444722-95-6),Olomoucine, ON-108600 (Onconova), ON-123300 (Onconova), Oxindole I,P-1446-05 (Piramal), P-276-00 (Piramal), palbociclib (Pfizer),PHA-767491 (CAS #845714-00-3), PHA-793887 (CAS #718630-59-2), PHA-848125(CAS #802539-81-7), Purvalanol A, Purvalanol B, R547 (CAS #741713-40-6),RO-3306 (CAS #872573-93-8), Roscovitine, SB-1317 (SBIO), SCH 900776 (CAS#891494-63-6), SEL-120 (Selvita), seliciclib (Cyclacel), SNS-032 (CAS#345627-80-7), SU9516 (CAS #377090-84-1), WHI-P180 (CAS #211555-08-7),pharmaceutically acceptable salts thereof, and combinations thereof.Preferably, the CDK inhibitor is selected from the group consisting ofdinaciclib, palbociclib, pharmaceutically acceptable salts thereof, andcombinationsthereof.

As used herein, a “poly ADP ribose polymerase (PARP) inhibitor” is asubstance that decreases the expression or activity of poly ADP ribosepolymerases (PARPs) or downstream proteins. Non-limiting examples ofpoly ADP ribose polymerase (PARP) inhibitors of the present inventioninclude PF01367338 (Pfizer, New York, N.Y.), olaparib (AstraZeneca,United Kingdom), iniparib (Sanofi-Aventis, Paris, France), veliparib(Abbott Laboratories, Abbott Park, Ill.), MK 4827 (Merck, White HouseStation, N.J.), CEP 9722 (Teva Pharmaceuticals, Israel), LT-673(Biomarin, San Rafael, Calif.), and BSI 401 (Sanofi-Aventis, Paris,France), pharmaceutically acceptable salts thereof, and combinationsthereof.

In a preferred embodiment, the chemotherapy comprises administering tothe human an agent selected from the group consisting of gemcitabine,taxol, adriamycin, ifosfamide, trabectedin, pazopanib, abraxane,avastin, everolimus, and combinations thereof.

As used herein, “radiotherapy” means any therapeutic regimen, that iscompatible with the C. novyi, e.g., C. novyi NT, treatment of thepresent invention and in which radiation is delivered to a subject,e.g., a human, for the treatment of cancer. Radiotherapy can bedelivered to, e.g., a human subject, by, for example, a machine outsidethe body (external-beam radiation therapy) or a radioactive materialinside the body (brachytherapy, systemic radiation therapy).

External-beam radiation therapy includes, but is not limited to,3-dimensional conformal radiation therapy, intensity-modulated radiationtherapy, image-guided radiation therapy, tomotherapy, stereotacticradiosurgery, stereotactic body radiation therapy, proton therapy, andother charged particle beam therapies, such as electron beam therapy.External-beam radiation therapies are widely used in cancer treatmentand are well known to those of skill in the art.

Brachytherapy means radiotherapy delivered by being implanted in, orplaced on, a subject's body. Brachytherapy includes, but is not limitedto, interstitial brachytherapy, intracavitary brachytherapy, andepiscleral brachytherapy. Brachytherapy techniques are also widely usedin cancer treatment and are well known to those of skill in the art.

Systemic radiation therapy means radiotherapy delivered by injection toor ingestion by a subject. One example of systemic radiation therapy isradioiodine therapy. Radioiodine is a radiolabeled iodine molecule thatis safe and effective for use in a subject, such as, e.g., a human.Non-limiting examples of radioiodine according to the present inventionmay be selected from the group consisting of ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, andcombinations thereof. Preferably, the radioiodine is ¹³¹I.

As used herein, “immunotherapy” means any anti-cancer therapeuticregimen that is compatible with the C. novyi, e.g., C. novyi NT,treatment of the present invention and that uses a substance that altersthe immune response by augmenting or reducing the ability of the immunesystem to produce antibodies or sensitized cells that recognize andreact with the antigen that initiated their production. Immunotherapiesmay be recombinant, synthetic, or natural preparations and includecytokines, corticosteroids, cytotoxic agents, thymosin, andimmunoglobulins. Some immunotherapies are naturally present in the body,and certain of these are available in pharmacologic preparations.Examples of immunotherapies include, but are not limited to, granulocytecolony-stimulating factor (G-CSF), interferons, imiquimod and cellularmembrane fractions from bacteria, IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7,and synthetic cytosine phosphate-guanosine (CpG).

In one preferred embodiment, the immunotherapy comprises administeringto the human an immune checkpoint inhibitor. As used herein, an “immunecheckpoint inhibitor” means a substance that blocks the activity ofmolecules involved in attenuating the immune response. Such moleculesinclude, for example, cytotoxic T-lymphocyte-associated antigen 4(CTLA-4) and programmed cell death protein 1 (PD-1). Immune checkpointinhibitors of the present invention include, but are not limited to,ipilimumab (Bristol-Myers Squibb), tremelimumab (Pfizer), MDX-1106(Medarex, Inc.), MK3475 (Merck), CT-011 (CureTech, Ltd.), AMP-224(Ampmmune), MDX-1105 (Medarex, Inc.), IMP321 (Immutep S.A.), and MGA271(Macrogenics).

In an additional aspect of this embodiment, the C. novyi, e.g., C. novyiNT, therapy of the present invention is effective against, e.g., solidtumors that are resistant to a therapy selected from the groupconsisting of chemotherapy, radiation therapy, immunotherapy, andcombinations thereof.

In another aspect of this embodiment, the solid tumor is refractory tostandard therapy or the solid tumor is without an available standardtherapy, yet the C. novyi, e.g., C. novyi NT, therapy of the presentinvention is effective against such a tumor.

As used herein, “resistant” and “refractory” are used interchangeably.Being “refractory” to a therapy means that the prior therapy ortherapies has/have reduced efficacy in, e.g., treating cancer or killingcancer cells, compared to the same subject prior to becoming resistantto the therapy.

As used herein, the term “standard therapy” means those therapiesgenerally accepted by medical professionals as appropriate for treatmentof a particular cancer, preferably a particular solid tumor. Standardtherapies may be the same or different for different tumor types.Standard therapies are typically approved by various regulatoryagencies, such as, for example, the U.S. Food and Drug Administration.

In a further aspect of this embodiment, the method induces a potentlocalized inflammatory response and an adaptive immune response in thehuman.

As used herein, an “inflammatory response” is a local response tocellular damage, pathogens, or irritants that may include, but is notlimited to, capillary dilation, leukocytic infiltration, swelling,redness, heat, itching, pain, loss of function, and combinationsthereof.

As used herein, an “adaptive immune response” involves B and T cells ofa subject's immune system. Upon exposure to a pathogenic substance, forexample, a cancer cell, B cells may produce antibodies againstpathogenic antigens on the pathogenic substance, and T cells may becomeable to target pathogens for eventual destruction. Certain populationsof B and T cells, specific for a given antigen, are retained by theimmune system and are called upon in the event of subsequent exposure tothe pathogenic antigen. An adaptive immune response is thus durable, andprovides a host subject's immune system with the continual ability torecognize and destroy a given pathogenic antigen-presenting pathogen.

Another embodiment of the present invention is a method for debulking asolid tumor present in a human. This method comprises administeringintratumorally to the human a unit dose of C. novyi, preferably C. novyiNT, CFUs comprising about 1×10³-1×10⁷ CFUs suspended in apharmaceutically acceptable carrier or solution.

As used herein, “debulking” a solid tumor means to reduce the size of orthe number of cancer in a solid tumor. Such a procedure is palliativeand may be used to enhance the effectiveness of the treatments,including radiation therapy, chemotherapy, or amputation. In thisembodiment, solid tumors are as set forth above. Preferably, the solidtumor is selected from the group consisting of soft tissue sarcoma,hepatocellular carcinoma, breast cancer, pancreatic cancer, andmelanoma. More preferably, the solid tumor is a leiomyosarcoma, such asa retroperitoneal leiomyosarcoma.

An additional embodiment of the present invention is a method fordebulking a solid tumor present in a human. This method comprisesadministering intratumorally to the human one to four cycles of a unitdose of C. novyi NT spores comprising about 1×10⁴ spores per cycle, eachunit dose of C. novyi NT being suspended in a pharmaceuticallyacceptable carrier or solution. In this embodiment, the types of solidtumors are as set forth above. Preferably, the solid tumor is selectedfrom the group consisting of soft tissue sarcoma, hepatocellularcarcinoma, breast cancer, pancreatic cancer, and melanoma.

A further embodiment of the present invention is a method for treatingor ameliorating an effect of a solid tumor present in a human. Thismethod comprises administering intratumorally to the human one to fourcycles of a unit dose of C. novyi NT spores comprising about 1×10⁴spores per cycle, each unit dose of C. novyi NT spores being suspendedin a pharmaceutically acceptable carrier or solution. Various types ofsolid tumors are as set forth above. Preferably, the solid tumor isselected from the group consisting of soft tissue sarcoma,hepatocellular carcinoma, breast cancer, pancreatic cancer, andmelanoma.

Another embodiment of the present invention is method for ablating asolid tumor present in a human. This method comprises administeringintratumorally to the human a unit dose of C. novyi, preferably C. novyiNT, CFUs comprising about 1×10³-1×10⁷ CFUs suspended in apharmaceutically acceptable carrier or solution, wherein the tumor isablated leaving a margin of normal tissue.

As used herein, “ablating” a solid tumor means that the process removesall of the solid tumor. In this process, after carrying out thetreatment, a margin of normal tissue is left surrounding the area wherethe tumor once resided. In this embodiment, the types of solid tumorsare as set forth above. Preferably, the solid tumor is a sarcoma. Morepreferably, the solid tumor is a leiomyosarcoma, such as aretroperitoneal leiomyosarcoma.

A further embodiment of the present invention is a unit dose of C. novyiCFUs. This unit dose comprises about 1×10³-1×10⁷ CFUs in apharmaceutically acceptable carrier or solution, which is effective fortreating or ameliorating an effect of a solid tumor present in a human.As set forth above, the C. novyi CFUs may be in vegetative and sporeforms.

In one aspect of this embodiment, the C. novyi is C. novyi NT.Preferably, the unit dose comprises about 1×10⁴-1×10⁷ C. novyi NTspores, such as about 1×10⁶-1×10⁷ C. novyi NT spores, in apharmaceutically acceptable carrier or solution. Preferably, the unitdose comprises about 1×10⁴ C. novyi NT spores in a pharmaceuticallyacceptable carrier or solution.

An additional embodiment of the present invention is a kit for treatingor ameliorating an effect of a solid tumor present in a human. This kitcomprises a unit dose of C. novyi CFUs comprising about 1×10³-1×10⁷ CFUsin a pharmaceutically acceptable carrier or solution and instructionsfor use of the kit. The kit may be divided into one or more compartmentsand may have one or more containers for the various reagents. The kitmay be further adapted to support storage and shipment of eachcomponent.

In one aspect of this embodiment, the kit further comprises one or moreantibiotics, which are effective to treat or alleviate an adverse sideeffect caused by the C. novyi CFUs. The CFUs may be in vegetative orspore forms. Suitable antibiotics are as set forth above. Preferably,the kit further comprises 1-4 unit doses of the C. novyi for carryingout 1-4 treatment cycles.

In another aspect of this embodiment, the C. novyi is C. novyi NT.Preferably, the unit dose comprises about 1×10⁴-1×10⁷ C. novyi NTspores, such as about 1×10⁶-1×10⁷ C. novyi NT spores, or about 1×10⁴ C.novyi NT spores, in a pharmaceutically acceptable carrier or solution.Also preferably, the kit further comprises 1-4 unit doses of the C.novyi NT spores for carrying out 1-4 treatment cycles.

Another embodiment of the present invention is a method formicroscopically precise excision of tumor cells in a human. This methodcomprises administering intratumorally to the human a unit dose of C.novyi NT colony forming units (CFUs) comprising about 1×10³-1×10⁷ CFUssuspended in a pharmaceutically acceptable carrier or solution.

As used herein, “microscopically precise excision” means elimination ofa target tissue in a subject, for example, a pathogenic tissue, saidelimination being essentially specific, at the cellular level, for thepathogenic tissue while causing minimal or no harm to nearby “healthy”tissue. Elimination of a target tissue may be, but is not limited to,apoptosis, necrosis, and cell lysis. This embodiment may be accomplishedby precision delivery of, e.g., the C. novyi NT sprores of the inventionvia CT-guided intratumoral injection using, e.g., a multi-prongeddelivery device, such as a multi-pronged needle.

In the present invention, the C. novyi spores, such as the C. novyi NTspores, are delivered to the subject, e.g., human patient,intratumorally in any medically appropriate manner. For example, C.novyi NT spores may be delivered via a single needle used at one or moresites on a tumor. Alternatively, a multi-tined delivery vehicle, such asa multi-tined needle, may be used to deliver, e.g., C. novyi NT spores,to a tumor. Delivery of, e.g., the spores may be to the same or multipledepths at one or more sites of the tumor. The selected delivery vehiclesmay be operated manually or controlled electronically. The deliveryvehicles may be positioned and/or repositioned on or within a tumormanually or via a remote controlled device and visualization of theinjection site may be augmented using various imaging techniques knownin the art, such as CT imaging. Multi-tined delivery vehicles that maybe used in the present invention include those disclosed in, e.g.,McGuckin, Jr. et al., U.S. Pat. Nos. 6,905,480 and 7,331,947, which areincorporated herein by reference.

A further embodiment of the present invention is a method for treatingor ameliorating an effect of a solid tumor that has metastasized to oneor more sites in a human. This method comprises administeringintratumorally to the human a unit dose of C. novyi NT colony formingunits (CFUs) comprising at least about 1×10³-1×10⁷ CFUs suspended in apharmaceutically acceptable carrier or solution. Preferably, at leastone site of metastasis is distal to the original solid tumor.

As used herein, “metastasis” and grammatical variations thereof mean thespread of pathogenic cells, i.e. tumor cells, from an original, primaryregion of the body, to a secondary region of the body. Metastasis may beregional or distal, depending on the distance from the original primarytumor site. Whether a metastasis is regional or distal may be determinedby a physician. For example, a breast cancer that has spread to thebrain is distal, whereas the spread of breast cancer cells to under armlymph nodes is regional.

In the present invention, an “effective amount” or a “therapeuticallyeffective amount” of a compound or composition disclosed herein is anamount of such compound or composition that is sufficient to effectbeneficial or desired results as described herein when administered to asubject. Effective dosage forms, modes of administration, and dosageamounts are as disclosed herein or as modified by a medicalprofessional. It is understood by those skilled in the art that thedosage amount will vary with the route of administration, the rate ofexcretion, the duration of the treatment, the identity of any otherdrugs being administered, the age and size of the patient, and likefactors well known in the arts of medicine. In general, a suitable doseof a composition according to the invention will be that amount of thecomposition, which is the lowest dose effective to produce the desiredeffect. The effective dose of a composition of the present invention isdescribed above. Further, a composition of the present invention may beadministered in conjunction with other treatments.

The compositions of the invention comprise one or more activeingredients in admixture with one or more pharmaceutically-acceptablecarriers and, optionally, one or more other compounds, drugs,ingredients and/or materials. Regardless of the route of administrationselected, the agents/compounds of the present invention are formulatedinto pharmaceutically-acceptable unit dosage forms by conventionalmethods known to those of skill in the art. See, e.g., Remington, TheScience and Practice of Pharmacy (21^(st) Edition, Lippincott Williamsand Wilkins, Philadelphia, Pa.).

Pharmaceutically acceptable carriers or solutions are well known in theart (see, e.g., Remington, The Science and Practice of Pharmacy (21^(st)Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and TheNational Formulary (American Pharmaceutical Association, Washington,D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, andsorbitol), starches, cellulose preparations, calcium phosphates (e.g.,dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable carrier orsolution used in a unit dose according to the present invention must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the subject. Carriers orsolutions suitable for a selected dosage form and intended route ofadministration, e.g., IT, are well known in the art, and acceptablecarriers or solutions for a chosen dosage form and method ofadministration can be determined using ordinary skill in the art.

The unit doses of the invention may, optionally, contain additionalingredients and/or materials commonly used in pharmaceuticalcompositions. These ingredients and materials are well known in the artand include (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monostearate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

Liquid dosage forms include pharmaceutically-acceptable emulsions,microemulsions, liquids, and suspensions. The liquid dosage forms maycontain suitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, coloring, andpreservative agents. Suspensions may contain suspending agents.

Dosage forms for the intratumoral administration include solutions,dispersions, suspensions or emulsions, or sterile powders. The activeagent(s)/compound(s) may be mixed under sterile conditions with asuitable pharmaceutically-acceptable carrier.

Unit doses of the present invention may alternatively comprise one ormore active agents, e.g., C. novyi CFUs or C. novyi NT spores incombination with sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsuitable antioxidants, buffers, solutes which render the formulationisotonic with the blood of the intended recipient, or suspending orthickening agents. Proper fluidity can be maintained, for example, bythe use of coating materials, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.These compositions may also contain suitable adjuvants, such as wettingagents, emulsifying agents and dispersing agents. It may also bedesirable to include isotonic agents. In addition, prolonged absorptionof the injectable pharmaceutical form may be brought about by theinclusion of agents which delay absorption.

Intratumorally injectable depot forms may be made by formingmicroencapsulated matrices of the active ingredient in biodegradablepolymers. Depending on the ratio of the active ingredient to polymer,and the nature of the particular polymer employed, the rate of activeingredient release can be controlled. Depot injectable formulations arealso prepared by entrapping the active agent in liposomes ormicroemulsions which are compatible with body tissue.

As noted above, the formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampules and vials, and may bestored in a lyophilized condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the typedescribed above.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Combined Intravenous (IV) Dosing of C. novyi NT withRadiation

A study of a single IV dose of C. novyi NT spores in dogs withspontaneous tumors following treatment with external beam radiation wasperformed.

The manufacturing and final formulation of C. novyi NT spores wasperformed by the Johns Hopkins Development laboratory according to thefollowing process. C. novyi NT spores generated according to Dang etal., 2001. were inoculated into a rich sporulation medium and incubatedin an anaerobic chamber for 17-19 days at 37° C. Spores were purified bysequential continuous Percoll gradient centrifugation followed byextensive phosphate buffered saline washing. Spores were stored at 2-8°C. Spores were prepared prior to shipment, suspended in sterilephosphate buffered saline and diluted in 50 ml of 0.9% sodium chloride.

C. novyi NT spores were reconstituted in a 50 ml saline bag anddelivered overnight to the test site. The radiation dose wasapproximately 54 gy delivered over 20 fractions: 11 before C. novyi NTIV injection and 9 after injection. C. novyi NT spores were administeredas a single injection at a dose of 1×10⁹ spores/m², based on bodysurface area. The transfer of the spores to a syringe occurred on anabsorbent pad with an impervious backing. A 22 gauge needle with a 3-waystopcock attached was inserted into the bag. A male portion of a closedchemotherapy system (ONGUARD™, TEVA Medical Ltd.) was attached to a porton the stopcock. The complete contents were withdrawn from the bag intoa 60 cubic centimeter (cc) syringe to which was attached a femaleportion of the closed system. The spores were injected into each subjectover 15 minutes through an IV catheter to which was attached the maleend of the chemotherapy closed system. The infusion was followed by a 10cc saline flush. The subject was monitored closely for 6 hourspost-infusion as follows: vital signs, blood pressure, and oxygensaturation monitoring every 15 minutes for the first 60 minutes,followed by monitoring every 30 minutes for the next 60 minutes, thenevery 60 minutes for the next 120 minutes. Subsequent checks wereperformed every 60 minutes for a total of 6 hours.

Test subjects were hospitalized for the initial 3 weeks of treatment: 2weeks for radiation treatments and 1 week following C. novyi NT IVtreatment. Subsequent follow-up visits occurred up to 6 monthspost-treatment at month 1, 2, 3, and 6. See Tables 1 and 2 for sampletreatment schedules.

TABLE 1 Schedule of spore events Screen Day 1 (Prior to In-Patientstarting Monitoring for Day Day Month Month Month Month radiation 6Hours Post 8 ± 2 15 ± 2 1 ± 3 2 ± 3 3 ± 14 6 ± 14 therapy) Infusion Day2 Day 3 Day 4 Day 5 days days days days days days Informed Consent XMedical History X Physical Exam X X X X X X X X X X X X Vital Signs X XX X X X X X X X X X Chest x-Ray X   X ¹   X ¹   X ¹   X ¹   X ¹   X ¹Tumor fine needle X X X X X aspiration (FNA) for culture AbdominalUltrasound X   X ¹   X ¹   X ¹   X ¹   X ¹   X ¹ Extremity x-Ray X   X ¹  X ¹   X ¹   X ¹   X ¹   X ¹ (if indicated) Complete blood count X X XX X X X X X X (CBC), Prothrombin time/Partial thromboplastin time(PT/PTT), Chem, Urinalysis Research bloodwork² X X X X X X X X X X X XTumor measurements X X X X X X X X X and photographs Infuse C. novyi XNT spores Response X X X X X X Adverse Events (AEs) X X X X X X X X XCon Meds X X X X X X X X X X ¹ Chest x-ray and additional imaging asclinically indicated ²Research lab work includes plasma, serum, wholeblood pellet, and peripheral blood mononuclear cell collection (cellsfrom plasma collection)

TABLE 2 Calendar of treatments for combined radiation and C. novyi NT(Days) Monday Tuesday Wednesday Thursday Friday Saturday SundayRadiation Day 1 Rad Day 2 Rad Day 3 Rad Day 4 Rad Day 5 Rad Day 6 RadDay 7 Rad Day 8 Rad Day 9 Rad Day 10 Spore day 1 X Rad Day 11 Rad Day 12Rad Day 13 Rad Day 14 Spore Day 6 Spore Day 7 Infusion X Spore Day 2 A YSpore Day 3 X Spore Day 4 Y Spore Day 5 X Rad Day 15 Rad Day 16 Rad Day18 B Rad Day 19 B Rad Day 20 B Spore Day 13 Spore Day 14 Spore Day 8 X,Y Spore Day 9 Spore Day 10 Spore Day 11 Spore Day 12 Spore Day 15 X, YSpore Day 30 X, Y Via CT tumor Spore Day 90 X, Y Re-evaluation 60 dayspost rad Spore Day 180 X, Z A Radiation may be interrupted more than oneday but will be radiation Day 11 when re-started B Radiation will becompleted one of these days. X = CBC, Chem Profile, AST, PT/PTT,Research Blood Samples, Adverse Events (AEs), Concomitant Medications,Tumor Measurements, Photos Y = Research blood samples Z = Thoracicmetastasis Check and additional Imaging as Indicated Including AbdominalUltrasound

As of Sep. 10, 2012, five dogs were treated in this manner. Of the five,2 developed an abscess, 1 maintained stable disease, and 2 died or wereeuthanized. The two test subjects that developed an abscess werephotographed throughout treatment as shown in FIGS. 1A and 1B.

FIG. 1A depicts a canine osteosarcoma located on the right distalradius/ulna over the course of treatment. The test subject, Sasha,exhibited fever and swelling on day 3 and a burst abscess on day 6.Antibiotics were started on day 8 due to the open wound and later,necrotic bone and tissue were removed. Sasha completed 12 of the 19radiation treatments and, as of Sep. 10, 2012, was healing with stabledisease.

FIG. 1B also depicts a canine osteosarcoma located on the right distalradius/ulna over the course of treatment. The test subject, Sampson,exhibited fever and swelling on day 5. On day 6, the abscess was lancedand antibiotics were started. Sampson completed 14 of the 20 radiationtreatments and, as of Sep. 10, 2012, was healing with stable disease.

The other subjects, Chipper, Bailey, and Ruskin, exhibited varyingresults. Chipper presented with a squamous cell carcinoma of the leftmandible. Over the course of treatment, Chipper had swelling at thetumor site and received 20 of 20 radiation treatments. As of Sep. 10,2012, Chipper had stable disease.

Another subject, Bailey, presented with a soft tissue sarcoma of theleft axillary region. During treatment, Bailey died, having experiencedsepsis, acute renal failure, potential disseminated intravascularcoagulation, and cardiac arrest. However, necropsy showed all deadtissue inside the tumor, with no tumor cells.

The remaining subject, Ruskin, presented with an osteosarcoma of theright proximal humerus. During treatment, Ruskin had swelling of thetumor site and completed 20/20 radiation treatments. However, on day 30,the tumor site was producing large amounts of purulent material andRuskin was experiencing renal failure. The owner decided to euthanizewhen renal status did not improve. As of Sep. 10, 2012, necropsy resultswere still pending.

Example 2 IT-Injected C. novyi-NT Spores Specifically Target TumorTissue and Prolong Survival in Rats—Methods Cell Lines and TissueCulture

A rat F98 glioma cell line transfected with a luciferase construct vialentivirus was maintained in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% fetal bovine serum (FBS) and 1% penicillin andstreptomycin.

Rat Experiments

6 week old female F344 Fisher rats (weight 100-150 grams) were purchasedfrom the National Cancer Institute. For the implantation procedure,female F344 Fisher rats were anesthetized via intraperitoneal (IP)injection of ketamine hydrochloride (75 mg/kg; 100 mg/mL ketamine HCl;Abbot Laboratories), xylazine (7.5 mg/kg; 100 mg/mL Xyla-ject; PhoenixPharmaceutical, Burlingame, Calif.), and ethanol (14.25%) in a sterileNaCl (0.9%) solution. F98 glioma cells (2×10⁴) were stereotacticallyimplanted through a burr hole into the right frontal lobe located 3 mmlateral and 2 mm anterior to the bregma, as described before (Bai, etal., 2011). Tumor size was assessed via a Xenogen instrument with IPinjection of 8 mg/rat D-luciferin potassium salt at day 12 afterimplantation of the tumor cells. Subsequently, 3 million C. novyi-NTspores, produced as previously described (Dang, et al., 2001,Bettegowda, et al., 2006), were stereotactically injected into theintracranial tumor using the same coordinates as described above and therats were treated with 10 mg/kg/day of IP dexamethasone for the first 2days. Animals were observed daily for any signs of deterioration,lethargy, neurotoxicity, or pain in accordance with the Johns HopkinsAnimal Care and Use Guidelines. If symptoms of distress were present,supportive therapy with hydration and doxycycline (loading dose of 15mg/kg IP followed by 10 mg/kg every 12 hours as maintenance) wasinitiated and continued for a 7 day period. If symptoms persisted and/orresulted in debilitation, moribund animals were euthanized. Theeffectiveness of IT injected C. novyi-NT spores was evaluated byKaplan-Meyer survival curves, as well as remaining tumor burden on brainsections. For the latter, brains were collected postmortem, placed informaldehyde, and embedded in paraffin for additional pathologicalstudies. Gram-stained slides, counter-stained with safranin, andH&E-slides were obtained according to standard procedure guidelines.

Statistical Analyses

Kaplan-Meier survival curves were created and analyzed with a Mantel-Coxtest using GraphPad Prism v.5.00 (GraphPad Software, San Diego, Calif.).

Example 3 IT-Injected C. novyi-NT Spores Specifically Target TumorTissue and Prolong Survival in Rats—Results

Complete surgical excision of advanced gliomas is nearly alwaysimpossible and these tumors inexorably recur. Though this tumor typegenerally does not metastasize, there are no highly effective medicaltherapies available to treat it. Gliomas therefore seemed to represent atumor type for which local injection of C. novyi-NT spores could betherapeutically useful. To evaluate this possibility, F98 rat gliomacells were orthotopically implanted into 6-week old F433 Fisher rats,resulting in locally invasive tumors that were rapidly fatal (FIG. 2A).IT injection of C. novyi-NT spores into the tumors of these ratsresulted in their germination within 24 hours and a rapid fall inluciferase activity, an indicator of tumor burden, over 24-48 hours(FIGS. 2B and 2C). C. novyi-NT germination was evidenced by theappearance of vegetative forms of the bacteria. Strikingly, C. novyi-NTprecisely localized to the tumor, sparing adjacent normal cells only afew microns away (FIGS. 3A and 3B). Moreover, these vegetative bacteriacould be seen to specifically grow within and concomitantly destroyislands of micro-invasive tumor cells buried within the normal brainparenchyma (FIGS. 4A and 4B). This bacterial biosurgery led to asignificant survival advantage in this extremely aggressive murine model(FIG. 2A, P-value <0.0001).

Example 4 Canine Soft Tissue Sarcomas Resemble Human Tumors—MethodsGenomic DNA Isolation for Sequencing

Genomic DNA from dogs participating in the comparative study of IT C.novyi-NT spores was extracted from peripheral blood lymphocytes (PBLs)and formalin-fixed, paraffin-embedded tumor tissue using the QIAamp DNAmini kit (QIAGEN, Valencia, Calif.) according to the manufacturer'sprotocol.

Sequencing and Bioinformatic Analysis

Genomic purification, library construction, exome capture, nextgeneration sequencing, and bioinformatics analyses of tumor and normalsamples were performed at Personal Genome Diagnostics (PGDx, Baltimore,Md.). In brief, genomic DNA from tumor and normal samples werefragmented and used for Illumina TruSeq library construction (Illumina,San Diego, Calif.). The exonic regions were captured in solution usingthe Agilent Canine All Exon kit according to the manufacturer'sinstructions (Agilent, Santa Clara, Calif.). Paired-end sequencing,resulting in 100 bases from each end of the fragments, was performedusing a HiSeq 2000 Genome Analyzer (Illumina, San Diego, Calif.). Thetags were aligned to the canine reference sequence (CanFam2.0) using theEland algorithm of CASAVA 1.7 software (Illumina, San Diego, Calif.).The chastity filter of the BaseCall software of Illumina was used toselect sequence reads for subsequent analysis. The ELAND algorithm ofCASAVA 1.7 software (Illumina, San Diego, Calif.) was then applied toidentify point mutations and small insertions and deletions. Knownpolymorphisms recorded in dbSNP131 (CanFam2.0) were removed from theanalysis. Potential somatic mutations were filtered and visuallyinspected as described previously (Jones, et al., 2010).

Example 5 Canine Soft Tissue Sarcomas Resemble Human Tumors—Results

Preclinical animal studies of anticancer agents often do notrecapitulate the observed effects in people. In dogs, however,clinically used therapeutic agents induce similar toxicities and effectsto people (Paoloni, et al., 2008). Studies of investigational therapiesin dogs can represent a crucial bridge between preclinical animalstudies and human clinical studies. In particular, canine soft tissuesarcomas are an excellent model as they are common in many breeds ofdogs and have clinical and histopathologic features remarkably close tothose of human soft tissue sarcomas (Paoloni, et al., 2008, Vail, etal., 2000). However, while recent advances in genomics havesignificantly expanded our knowledge of cancer genetics in people,comparatively little is known about the genetic landscape of caninecancers. Therefore, to determine whether canine tumors were geneticallysimilar to those of humans, the exome of tumor and matched normal DNAfrom 11 dogs participating in the comparative study was sequenced (FIG.5). This analysis involved the interrogation of 30,194 nominal genescomprising 32.9 megabases (Mb) of DNA. Ten of the dogs had soft tissuesarcomas (six peripheral nerve sheath tumors) and one had achondroblastic osteosarcoma. On average, 15.7 gigabases (Gb) (range:8.1-23.3 Gb) of generated sequence were mapped to the genome, and 92.1%of bases in the targeted regions were covered by at least 10 uniquereads in the tumor DNA. Similarly, an average of 16.3 Gb (range:14.6-19.7 Gb) of sequence were mapped to the genome in normal DNA, with93.6% of targeted bases covered by at least ten unique reads. Averagecoverage for each targeted base in the tumor was 153-fold (range:73-227-fold) and was 152-fold in the matched normal samples (range:130-178-fold).

Using stringent analysis criteria, 156 somatic mutations and 28 somaticcopy number alterations among the 10 soft tissue sarcomas wereidentified (Table 3 and FIG. 6). The range of somatic mutations was 0 to95 with a mean of 14 per tumor. Mutation prevalence in the soft tissuesarcomas was low, averaging 0.47 per Mb (range: 0.00-2.89 per Mb).Excluding one sample outlier, with 95 somatic alterations, there was amean prevalence of 0.21 mutations per Mb (range: 0.00-0.61 per Mb) (FIG.5), similar to estimates of the mutation rate in human pediatricrhabdoid tumors (Lee, et al., 2012) and other soft tissue sarcomas(Joseph, et al., 2013). The most common type of somatic alteration was amissense mutation, with a preponderance of C to T (45.5%) and G to Atransitions (34.0%; Tables 4a and 4b).

TABLE 3 Somatic Alterations in Canine Sarcomas Sequence Context Amino(Posi- Acid tion of Nucleotide (protein) Muta- % Tu- (genomic) Positiontion In- Mu- Case mor Gene Gene Transcript Position of of Mutationdicated tant ID Type Symbol Description Accession Mutation Mutation TypeConsequence by “N”) Reads 04- STS CCDC61 coiled-coil ENSCAFT000chr1_112524782- NA Substitution Splice site  CCCTANC 0.41 R03 domain00006986 112524782_C_T donor TGGG containing 61 (SEQ ID NO: 1) FAM83Bfamily with ENSCAFT000 chr12_25277449- 68V > F SubstitutionNonsynonymous AAAACNT 0.39 sequence 00003643 25277449_G_T   coding CCAGsimilarity 83, (SEQ ID member B NO: 2) Novel uncharacterized ENSCAFT000chr23_3005035- 32N > I Substitution Nonsynonymous GGTCANT 0.34 Geneprotein 00006899 3005035_T_A   coding ATTA (SEQ ID NO: 3) Noveluncharacterized ENSCAFT000 chr20_55267898- 323R > X SubstitutionNonsense AGGAGNG 0.17 Gene protein 00028936 55267898_C_ ACGC (SEQ IDNO: 4) NUP210 nucleoporin ENSCAFT000 chr20_6644043- 1627P > TSubstitution Nonsynonymous GCCCGN 0.38 210kDa 00007053 6644043_G_Tcoding GATGG (SEQ ID NO: 5) PLMN Plasminogen ENSCAFT000 chr1_52549843-598G > E Substitution Nonsynonymous CGCACNC 0.28 Plasmin heavy 0000117952549843_C_T coding ACCT chain A Plasmin (SEQ ID light chain B NO: 6)UFSP2 UFM1-specific ENSCAFT000 chr16_48180970- 271L > R SubstitutionNonsynonymous TTACCNC 0.61 peptidase 2 00012105 48180970_T_G coding AATC(SEQ ID NO: 7) ZNFX1 zinc finger, ENSCAFT000 chr24_38909185- 1195I > LSubstitution Nonsynonymous AACAANG 0.34 NFX1-type 00018115 38909185_T_Gcoding TCAT containing 1 (SEQ ID NO: 8) 16- STS ANKRD1 ankyrin repeatENSCAFT000 chr5_67220009- NA Substitution Splice site  CCGTGNT 0.19 R031 domain 11 00031567 67220009_G_A donor GAGT (SEQ ID NO: 9) TMEM13transmembrane ENSCAFT000 chr26_7467030- 198G > D SubstitutionNonsynonymous ACAAGNC 0.18 2B protein 132B 00011029 7467030_C_T codingGGCC (SEQ ID NO: 10) 16- STS CAPN6 calpain 6 ENSCAFT000 chrX_87423838-433R > H Substitution Nonsynonymous ATCTGCG 0.45 R02 0002887287423838_C_T coding GTTC (SEQ ID NO: 11) CNGB3 cyclic ENSCAFT000chr29_35801978- 451R > X Substitution Nonsense GATTCGG 0.22 nucleotide-00014134 35801978_G_A AAGT gated cation (SEQ ID channel beta-3 NO: 12)Novel uncharacterized ENSCAFT000 chr4_69847894- 352Y > X SubstitutionNonsense ACCTACT 0.11 gene protein 00035928 69847894_C_G TTGA (SEQ IDNO: 13) PLAC8L1 PLAC8-like 1 ENSCAFT000 chr2_43368179- 99C > YSubstitution Nonsynonymous TGTCACA 0.2 00010364 43368179_C_T coding CTCA(SEQ ID NO: 14) 11- STS AIDA axin  ENSCAFT000 chr38_19939874- 258F > SSubstitution Nonsynonymous AAGCANA 0.25 R04 interactor, 0002148619939874_A_G coding GCAC dorsalization (SEQ ID associated NO: 15) BRWD3bromodomain ENSCAFT000 chrX_65189965- 275S > A SubstitutionNonsynonymous AGTTGNT 0.7 and WD repeat 00027493 651E19965_A _C codingGGAC domain (SEQ ID containing 3 NO: 16) Novel uncharacter- ENSCAFT000chrX_58551749- 104K > R Substitution Nonsynonymous CCTGANG 0.17 geneized 00027037 5851749_A_G coding AATT protein (SEQ ID NO: 17) 11- STS-AFAP1L1 actin filament ENSCAFT000 chr4_62838379- 4255 > F SubstitutionNonsynonymous TCTTGNA 0.25 R02 PNST associated 00029078 6288379_G_Acoding GAAG protein 1- (SEQ ID like 1 NO: 18) ATP7B copper- ENSCAFT000chr22_3134952- 288K > Q Substitution Nonsynonymous ACCCANA 0.2transporting 00006859 3134952 _A _C coding GATG ATPase 2 (SEQ ID NO: 19)C11orf63 chromosome 11 ENSCAFT000 chr5_14445155- 55S > P SubstitutionNonsynonymous CTGGGNC 0.18 open reading 00018556 14445155_A_G codingTTAC frame 63 (SEQ ID NO: 20) FIP1L1 FIP1 like 1(S. ENSCAFT000chr13_48967897- NA Deletion Frameshift AGGTANA 0.4 cerevisiae) 0000322048967897_C_ GCAG (SEQ ID NO: 21) KRT23 keratin 23 ENSCAFT000chr9_25094298- 389K > M Substitution Nonsynonymous ATCGANG 0.25 (histone00025377 25094298_A_T coding TCAA deacetylase (SEQ ID inducible) NO: 22)MLL3 myeloid/lymphoi ENSCAFT000 chr16_18937990- 3177QQ DeletionIn-frame  GCTGTNG 0.11 d or mixed- 00007959 1893T992_TGC_  > Q deletionCTGC lineage (SEQ ID leukemia 3 NO: 23) MUC5AC mucin 5B, ENSCAFT000chr18_48561759- 3305G > S Substitution Nonsynonymous AGACANG 0.12oligomeric 00015796 485671759_G_A coding CCCC mucus/gel- (SEQ ID formingNO: 24) Novel uncharacterized ENSCAFT000 chr14_61936959- NA InsertionFrameshift CGGTCNC 0.16 gene protein 00036128 61936959_T CCAG (SEQ IDNO: 25) OR52N1 olfactory ENSCAFT000 chr21_32133356- 239A > TSubstitution Nonsynonymous GAAGGNC 0.28 receptor,  00010210 32133356_C_Tcoding TTCT family 52,  (SEQ ID subfamily N, NO: 26) member 1 PREX1phosphatidylino ENSCAFT000 chr24_38467733- 96R > H SubstitutionNonsynonymous AGGCGN 0.29 sito1-3,4,5- 00017540 38467733_C_T codingGCACA trisphosphate- (SEQ ID dependent Rac NO: 27) exchange factor 1PRPF39 PRP39 pre- ENSCAFT000 chr8_25550886- NA Deletion FrameshiftGAAGANT 0.24 mRNA 00022300 25550886_T_ TTGG processing (SEQ ID factor 39NO: 28) homolog Q6W651 uncharacterized ENSCAFT000 chr9_50634661-3105 > T Substitution Nonsynonymous TTTGGNT 0.27 protein 0003069750634661_A_T coding TTAT (SEQ ID NO: 29) TENM2 teneurin ENSCAFT000chr4_46714792- 364R > H Substitution Nonsynonymous TTCGGNG 0.21transmembrane 00027184 46714792_C_T coding GCGG protein 2 (SEQ IDNO: 30) ZNF641 zinc finger ENSCAFT000 chr27_9390690- 363P > SSubstitution Nonsynonymous CCCCCNC 0.26 protein 641 00014313 9390690_C_Tcoding AGTG (SEQ ID NO: 31) 11- STS- ACTN2 actinin,  ENSCAFT000chr4_6385028- 90G > E Substitution Nonsynonymous TTTTTNCT 0.24 R01 PNSTalpha 2 00017321 6385028_C_T coding CGG (SEQ ID NO:  32) GPR139G protein- ENSCAFT000 chr6_28316728- 132P > L Substitution NonsynonymousCCACCNG 0.27 coupled 00028634 28316728_C_T coding CTCA receptor 139(SEQ ID NO: 33) KCNJ16 potassium ENSCAFT000 chr9_19566120- 5G > CSubstitution Nonsynonymous ATTACNG 0.26 inwardly- 00017085 19566120_G_Tcoding CAGC rectifying (SEQ ID channel, NO: 34) subfamily J, member 16KCNJ5 potassium ENSCAFT000 chr5_8746471- 116G > R SubstitutionNonsynonymous ATCACNC 0.32 inwardly- 00016271 8746471_C_G coding CGGArectifying (SEQ ID channel, NO: 35) subfamily J, member 5 04- STS-A1ILJ0 serpin  ENSCAFT000 chr8_66432888- 194D > N SubstitutionNonsynonymous GACATNC 0.42 R08 PNST peptidase 00036554 66432888_C_Tcoding TCTA inhibitor, (SEQ ID clade NO: 36) A (alpha-1 antiproteinase,antitrypsin), member 1 precursor AASS aminoadipate- ENSCAFT000chr14_62956632- 66G > S Substitution Nonsynonymous AATGCNA 0.62semialdehyde 00005673 62956632_C_T coding CCAG synthase (SEQ ID NO: 37)ABCB10 ATP-binding ENSCAFT000 chr4_12734254- 495R > C SubstitutionNonsynonymous CAGCTNG 0.47 cassette, sub- 00019279 12734254_C_T codingCCCA family B (SEQ ID (MDR/TAP), NO: 38) member 10 ACTL9 actin-like 9ENSCAFT000 chr20_56179685- 363P > S Substitution Nonsynonymous GGGGGN0.37 00029470 56179685_G_A coding CAGGC (SEQ ID NO: 39) ADAM7 ADAMENSCAFT000 chr25_35952270- 473E > K Substitution Nonsynonymous CACTTNA0.31 metallopeptidas 00014408 35952270 C T coding GGAA e domain 7(SEQ ID NO: 40) ADCYAP adenylate ENSCAFT000 chr14_46708954- 4485 > FSubstitution Nonsynonymous GGGCTNC 0.63 1R1 cyclase 0000501846708954_C_T coding TTCC activating (SEQ ID polypeptide 1 NO: 41)(pituitary) receptor type I ALDH7A aldehyde ENSCAFT000 chr11_18836811-523T > I Substitution Nonsynonymous TGATANT 0.3 1 dehydrogenase 0000090418836811_G_A coding ACTA 7 family, (SEQ ID member A1 NO: 42) ANKLE1ankyrin repeat ENSCAFT000 chr20_48444251- 74Q > X Substitution NonsenseCTCCTNG 0.27 and LEM 00024464 48444251_G_A TCTC domain (SEQ IDcontaining 1 NO: 43) ARMC9 armadillo  ENSCAFT000 chr25_46161506-296T > I Substitution Nonsynonymous TTCAANC 0.29 repeat 0001750846161506_C_T coding ATGT containing 9 (SEQ ID NO: 44) ASPM AbnormalENSCAFT000 chr7_8578487- 1156L > F Substitution Nonsynonymous CATTTNTT0.2 spindle-like 00018114 8578487_C_T coding TGC (SEQ microcephaly-ID NO:  associated 45) protein homolog ATP13A1 ATPase type ENSCAFT000chr20_46627633- 6335 > F Substitution Nonsynonymous AATGTNC 0.2 13A100022481 46627633_C_T coding GTGC (SEQ ID NO: 46) ATP2B3 ATPase, Ca++ENSCAFT000 chrX_124404772- 22P > L Substitution Nonsynonymous GGCCCNC0.19 transporting, 00030531 124404772_C_T coding CATG plasma (SEQ IDmembrane 3 NO: 47) B6EY10 tryptophan 5- ENSCAFT000 chr21_43753174-98R > Q Substitution Nonsynonymous ATTTTNG 0.47 hydroxylase 1 0001448543753174_C_T coding GGAC (SEQ ID NO: 48) BCAR1 breast cancer ENSCAFT000chr5_78491554- 150P > S Substitution Nonsynonymous AGATGNC 0.28anti-estrogen 00031962 78491554_C_T coding CCAT resistance 1 (SEQ IDNO: 49) BOD1L1 biorientation  ENSCAFT000 chr3_69317598- 2128P > SSubstitution Nonsynonymous AACTCNC 0.29 of 00024431 69317598_C_T codingTGCG chromosomes in (SEQ ID cell NO: 50) division 1- like 1 BRDTbromodomain, ENSCAFT000 chr6_59977191- 874E > K SubstitutionNonsynonymous ATTTTNTT 0.5 testis-specific 00032118 59977191_C_T codingGAA (SEQ ID NO:  51) BRE brain and ENSCAFT000 chr17_25386278- 372Q > HSubstitution Nonsynonymous AACCANC 0.36 reproductive 0000839725386278_G_T coding CTTC organ- (SEQ ID expressed NO: 52) (TNFRSF1Amodulator) C11orf80 chromosome 11 ENSCAFT000 chr18_53566794- 206P > LSubstitution Nonsynonymous TCAGANG 0.45 open reading 0001946053566794_G_A coding CAGA frame 80 (SEQ ID NO: 53) C1orf168 chromosome 1ENSCAFT000 chr5_55715053- 219T > I Substitution Nonsynonymous AGAAANC0.26 open reading 00030112 55715053_C_T coding CCTC frame 168 (SEQ IDNO: 54) C6orf211 chromosome 6 ENSCAFT000 chr1_44848305- 38R > XSubstitution Nonsense TGCATNG 0.32 open reading 00000674 44848305_C_TACAT frame 211 (SEQ ID NO: 55) CABP2 calcium binding ENSCAFT000chr18_52987478- 67G > E Substitution Nonsynonymous AGTGGNG 0.35protein 2 00018054 52987478 G A _ _ coding CCGG (SEQ ID NO: 56) CEP250centrosomal ENSCAFT000 chr24_27405113- 550L > F SubstitutionNonsynonymous TCATTNTT 0.6 protein 250kDa 00012850 27405113_C_T codingCGG (SEQ ID NO:  57) CSMD1 CUB and Sushi ENSCAFT000 chr16_58244318-15515 > F Substitution Nonsynonymous TCTGGNA 0.48 multiple 0001388558244318_G_A coding ATGG domains 1 (SEQ ID NO: 58) CSMD2 CUB and SushiENSCAFT000 chr15_11028241- 7285 > L Substitution Nonsynonymous GACTTNG0.18 multiple 00005882 11028241_C_T coding CCCA domains 2 (SEQ IDNO: 59) DCDC2 doublecortin ENSCAFT000 chr35_25388917- 192G > ESubstitution Nonsynonymous GTTTTNC 0.54 domain 00016283 25388917_C_Tcoding TTCT containing 2 (SEQ ID NO: 60) DNMT3B DNA (cytosine-ENSCAFT000 chr24_25068698- 615 > F Substitution Nonsynonymous ATTGTNC0.26 5-)- 00011678 25068698_C_T coding AAGA methyltransfera (SEQ IDse 3 beta NO: 61) EMR2 EGF-like ENSCAFT000 chr20_50969425- 755 > NSubstitution Nonsynonymous GGCTGNT 0.43 module- 00025982 50969425_C_Tcoding GAAG containing (SEQ ID mucin-like NO: 62) hormonereceptor-like 2 precursor EXOC3L exocyst ENSCAFT000 chr5_85189666-539R > K Substitution Nonsynonymous GGTGANA 0.46 1 complex 0003245585189666_G_A coding GTCC component 3- (SEQ ID like 1 NO: 63) FCRLBFc receptor- ENSCAFT000 chr38_23962108- 21A > S SubstitutionNonsynonymous GGCTGNC 0.14 A like 00020702 23962108_C_A coding CAGA(SEQ ID NO: 64) FLRT1 fibronectin ENSCAFT000 chr18_55953743- 616G > DSubstitution Nonsynonymous CGGGGN 0.31 leucine rich 0002338555953743_C_T coding CCCGG transmembrane (SEQ ID protein 1 NO: 65) FMR1fragile X  ENSCAFT000 chrX_119344462- 331E > K SubstitutionNonsynonymous CCAAGNA 0.24 mental 00030311 11944462_G_A coding AATTretardation 1 (SEQ ID NO: 66) FMR1 fragile X  ENSCAFT000 chrX_119344481-3375 > F Substitution Nonsynonymous AAATTNC 0.2 mental 0003031111944481_C_T coding CTAC retardation 1 (SEQ ID NO: 67) FSCN3fascin homolog ENSCAFT000 chr14_11685668- 310R > C SubstitutionNonsynonymous TGCACNA 0.48 3, actin-bund- 00002697 11685668_G_A codingAGCT ling protein, (SEQ ID testicular NO: 68) (Strongylocentrotus purpuratus) FUT9 Alpha-(1,3)- ENSCAFT000 chr12_57775088- 331E > KSubstitution Nonsynonymous TTTGGNA 0.28 fucosyltrans- 0000550757775088_G_A coding ATCA ferase (SEQ ID NO: 69) FXYD3 FXYD domainENSCAFT000 chr1_120363321- NA Substitution Splice site  TCTCANC 0.88containing ion 00011413 12063321_C_T donor ATAG transport (SEQ IDregulator 3 NO: 70) GPR126 G protein- ENSCAFT000 chr1_37098753- 4155 > FSubstitution Nonsynonymous AATTTNC 0.24 coupled 00000457 37098753_C_Tcoding ATAG receptor 126 (SEQ ID NO: 71) GPR128 G protein- ENSCAFT000chr33_10191962- 34R > W Substitution Nonsynonymous AAGGANG 0.33 coupled00014844 1019-1962_C_T coding GAGG receptor 128 (SEQ ID NO: 72) GPR82G protein- ENSCAFT000 chrX_36056596- 213S > L Substitution NonsynonymousATTTTNAT 0.32 coupled 00022877 36056596_C_T coding TTT (SEQ receptor 82ID NO:  73) GRM6 glutamate ENSCAFT000 chr11_5596380- 523P > LSubstitution Nonsynonymous CCTCCNC 0.53 receptor, 00000509 5596-380_C_Tcoding TGTG metabotropic 6 (SEQ ID NO: 74) GSX1 GS homeobox 1 ENSCAFT000chr25_14841844- NA Substitution Splice site GCTGTNT 0.36 0001087014841844_C_T acceptor GGAG (SEQ ID NO: 75) GTF2I general ENSCAFT000chr6_8807549- 145Q > X Substitution Nonsense AGACTNA 0.43 transcription00038018 8807549_G_A TCTC factor Ili (SEQ ID NO: 76) HDAC8 histoneENSCAFT000 chrX_59408793- 3595 > F Substitution Nonsynonymous GGGAANA0.71 deacetylase 8 00027174 59408793_G_A coding GAAG (SEQ ID NO: 77)HECTD4 HECT domain ENSCAFT000 chr26_12845851- 541R > Q SubstitutionNonsynonymous CTTCCNG 0.38 containing E3 00014076 12845851_C_T codingCTTG ubiquitin  (SEQ ID protein NO: 78) ligase 4 K1C10 keratin, type IENSCAFT000 chr9_25194405- 316E > K Substitution Nonsynonymous AATACNA0.3 cytoskeletal 10 00025391 25194405_G_A coding ACAA (SEQ ID NO: 79)KCNG3 potassium ENSCAFT000 chr17_37144629- 3665 > F SubstitutionNonsynonymous TGTTGNA 0.43 voltage-gated 00035514 37144629_G_A codingTGTT channel, (SEQ ID subfamily G, NO: 80) member 3 KIF25 kinesin familyENSCAFT000 chr1_58634208- 509E > K Substitution Nonsynonymous TGTCGNA0.33 member 25 00001345 58634208_G_A coding GCGC (SEQ ID NO: 81) LAMB2laminin, beta 2 ENSCAFT000 chr20_43058275- 1054P > L SubstitutionNonsynonymous GTGCCNG 0.38 (laminin S) 00018765 43058275_C_T coding TCCA(SEQ ID NO: 82) LIMK1 LIM domain ENSCAFT000 chr6_9274167- 222R > WSubstitution Nonsynonymous GATCCNG 0.6 kinase 1 00019799 9274167_G_Acoding TCTC (SEQ ID NO: 83) LY9 lymphocyte ENSCAFT000 chr38_24536297-263E > K Substitution Nonsynonymous CGACTNC 0.58 antigen 9 0002005624536297_C_T coding CCCA (SEQ ID NO: 84) MBD5 methyl-CpG ENSCAFT000chr19_53239621- 1189P > L Substitution Nonsynonymous TGGTCNA 0.32binding domain 00008917 53239621_C_T coding GCTA protein 5 (SEQ IDNO: 85) MLF1 myeloid ENSCAFT000 chr23_54989572- 164A > V SubstitutionNonsynonymous CCGAGNT 0.33 leukemia factor 00014162 54989572_C_T codingCATG 1 (SEQ ID NO: 86) NELL1 NEL-like 1 ENSCAFT000 chr21_46027895-105E > K Substitution Nonsynonymous CTGTCNA 0.24 (chicken) 0001591946027895_G_A coding ATGT (SEQ ID NO: 87) NF1 neurofibromin 1 ENSCAFT000chr9_44834512- 1933P > S Substitution Nonsynonymous CCACGNA 0.4800029545 44834512_G_A coding GTCA (SEQ ID NO: 88) Novel UncharacterizedENSCAFT000 chr27_39478508- 1291E > K Substitution Nonsynonymous GTTCTNA0.36 gene protein 00021819 39478508_G_A coding ACTA (SEQ ID NO: 89)Novel Uncharacterized ENSCAFT000 chr1_106460436- 314E > K SubstitutionNonsynonymous GGGAGNA 0.47 gene protein 00004310 106460436_G_A codingGAAA (SEQ ID NO: 90) Novel Uncharacterized ENSCAFT000 chr6_27157711-319M > I Substitution Nonsynonymous AAAATNA 0.39 gene protein 0002822227157711_C_T coding TGCA (SEQ ID NO: 91) Novel UncharacterizedENSCAFT000 chr8_56643270- 395R > C Substitution Nonsynonymous TAAACNA0.38 gene protein 00027418 56643270_G_A coding TCAG (SEQ ID NO: 92)Novel Uncharacterized ENSCAFT000 chr25_30547894- 397D > N SubstitutionNonsynonymous GGCATNA 0.31 gene protein 00012946 30547894_G_A codingTGGC (SEQ ID NO: 93) Novel Uncharacterized ENSCAFT000 chrX_115997637-6E > K Substitution Nonsynonymous CAATTNG 0.41 gene protein 00030235115997637_C_T coding CCAG (SEQ ID NO: 94) Novel UncharacterizedENSCAFT000 chr6_14378075- 734S > F Substitution Nonsynonymous TTTTGNA0.36 gene protein 00024549 14378075_G_A coding AATT (SEQ ID NO: 95)Novel Uncharacterized ENSCAFT000 chr1_116977163- 56E > X SubstitutionNonsense CACTTNG 0.17 gene protein 00009040 11g77163_C_A GAGC (SEQ IDNO: 96) NTN5 netrin 5 ENSCAFT000 chr1_110537423- 259W > X SubstitutionNonsense CTTCTNG 0.17 00006331 110E37423_ G_A AGGG (SEQ ID NO: 97)NUP210 nucleoporin ENSCAFT000 chr7_46057921- 287P > S SubstitutionNonsynonymous GATTTNC 0.25 L 210kDa-like 00027524 460E7921_C_T codingTCTG (SEQ ID NO: 98) NVL nuclear VCP- ENSCAFT000 chr7_43088033- 7835 > LSubstitution Nonsynonymous CTACTNG 0.16 like 00025949 430E8033_C_Tcoding TGAG (SEQ ID NO: 99) OLFM4 olfactomedin 4 ENSCAFT000chr22_13020301- 245Q > H Substitution Nonsynonymous GTTCANC 0.2600038323 13020301_G_C coding TCAA (SEQ ID NO: 100) OR11H4 olfactoryENSCAFT000 chr15_20603710- 352M > I Substitution Nonsynonymous GACATNA0.33 receptor,  00008634 2060-3710_G _A coding AATT family 11,  (SEQ IDsubfamily H, NO: 101) member 4 OR11L1 olfactory ENSCAFT000chr14_4576143- 164S > F Substitution Nonsynonymous GATTTNC 0.25receptor,  00039246 4576143_C_T coding AAGT family 11,  (SEQ IDsubfamily L, NO: 102) member 1 PEPB pepsin B ENSCAFT000 chr6_43778633-367D > N Substitution Nonsynonymous TGGGANA 0.14 precursor 0003138843778633_G_A coding TGTC (SEQ ID NO: 103) PHKA2 phosphorylase ENSCAFT000chrX_14879295- NA Substitution Splice site  ACTTANT 0.46 kinase, alpha 00020564 14879295_C_T donor TTAT 2 (liver) (SEQ ID NO: 104) PKHD1polycystic ENSCAFT000 chr12_22675987- 13235 > L SubstitutionNonsynonymous TCACTNA 0.38 kidney and 00003416 22675987_G_A coding GTTGhepatic disease (SEQ ID 1 (autosomal NO: 105) recessive) PRDM2 PR domainENSCAFT000 chr2_86311966- 1366P > S Substitution Nonsynonymous GGACGNC0.31 containing 2, 00025940 86311966_G_A coding AGCG with ZNF (SEQ IDdomain NO: 106) PTPRO protein  ENSCAFT000 chr27_34189070- 309E > KSubstitution Nonsynonymous TTTTTNC 0.57 tyrosine 00020369 34189070_C_Tcoding GTCT phosphatase, (SEQ ID receptor NO: 107) type, O PTPRZ1protein  ENSCAFT000 chr14_62891929- 1733L > P Substitution NonsynonymousTAAACNT 0.11 tyrosine 00005646 62891929_T_C coding GCAC phosphatase,(SEQ ID receptor- NO: 108) type, Z polypeptide 1 Q28302 UncharacterizedENSCAFT000 chr20_54398781- 202L > F Substitution Nonsynonymous AACTCNT0.34 protein 00035111 54398781_C_T coding CAAC (SEQ ID NO: 109) Q38IV3Multidrug ENSCAFT000 chr9_29903253- 761R > Q Substitution NonsynonymousCCAGCNA 0.47 resistance 00027259 29903253_G_A coding CAGC protein 3(SEQ ID NO: 110) Q8HYR2 Uncharacterized  ENSCAFT000 chr27_29388021-166I > F Substitution Nonsynonymous GAAATNT 0.59 protein 0001963329388021_A_T coding TATA (SEQ ID NO: 111) RCC2 regulator of ENSCAFT000chr2_83776440- 309P > L Substitution Nonsynonymous GGTCCNC 0.46chromosome 00024961 83776440_C_T coding CGGC condensation 2 (SEQ IDNO: 112) RP1 oxygen- ENSCAFT000 chr29_9140829- 1861E > K SubstitutionNonsynonymous AATCANA 0.3 regulated 00011204 9140829_G_A coding AAGAprotein 1 (SEQ ID NO: 113) RTKN2 rhotekin 2 ENSCAFT000 chr4_17382177-6025 > L Substitution Nonsynonymous GCCATNA 0.29 00020670 173E12177_G_Acoding TCTG (SEQ ID NO: 114) SAMD7 sterile alpha ENSCAFT000chr34_37539386- 369R > Q Substitution Nonsynonymous TCTTCNA 0.29motif domain 00023423 37539386_G_A coding AGCA containing 7 (SEQ IDNO: 115) SLAF1 Signaling ENSCAFT000 chr38_24663637- 2335 > LSubstitution Nonsynonymous GTCTTNG 0.53 lymphocytic 0001998224663637_C_T coding GGTG activation (SEQ ID molecule NO: 116) SLC47A2solute carrier ENSCAFT000 chr5_43495248- 83S > F SubstitutionNonsynonymous AGTTTNC 0.38 family 47, 00036298 43495248_C_T coding ATAGmember 2 (SEQ ID NO: 117) SULT4A sulfotrans- ENSCAFT000 chr10_24862764-72M > I Substitution Nonsynonymous TTGATNA 0.26 1 ferase 0003567424862764_G_A coding ACAT family 4A, (SEQ ID member 1 NO: 118) TAF7LTAF7-like RNA ENSCAFT000 chrX_78291782- 366E > K SubstitutionNonsynonymous CTTTTNAT 0.41 polymerase II, 00027954 78291782_C_T codingAAT (SEQ TATA box ID NO: binding protein 119) (TBP)- associatedfactor, 50kDa TBC1D1 TBC1 domain ENSCAFT000 chr10_16382190- 1765 > FSubstitution Nonsynonymous TGACTNT 0.3 5 family, member 0000073516382190_C_T coding CTTG 15 (SEQ ID NO: 120) TLR1 toll-like  ENSCAFT000chr3_76368607- 234W > X Substitution Nonsense GGATGNT 0.3 receptor00037196 76368607_G_A CTTA 1 precursor (SEQ ID NO: 121) TMEM74transmembrane ENSCAFT000 chr13_12451185- 61R > C SubstitutionNonsynonymous AGGGCNA 0.34 protein 74 00001114 12451185_G_A coding AGTT(SEQ ID NO: 122) TOM1 target of myb1 ENSCAFT000 chr10_31874137- 50V > GSubstitution Nonsynonymous GCATCNC 0.36 (chicken) 00002700 31874137_A_Ccoding CTCA (SEQ ID NO: 123) TRIM58 tripartite  ENSCAFT000chr14_4533386- 455T > R Substitution Nonsynonymous CGTTTNT 0.23 motif00001915 4533386_G_C coding TACA containing 58 (SEQ ID NO: 124) TRIM66tripartite  ENSCAFT000 chr21_35253035- 662L > F SubstitutionNonsynonymous TGGGANA 0.43 motif 00011106 35253035_G_A coding GGCGcontaining 66 (SEQ ID NO: 125) TTN titin ENSCAFT000 chr36_25212813-25277E >  Substitution Nonsynonymous ACTTTNTT 0.31 00022319 25212813_C_TK coding TAA (SEQ ID NO: 126) TTN titin ENSCAFT000 chr36_25208898-26582P >  Substitution Nonsynonymous GACCGNT 0.36 00022319 25208898_G_AS coding TCGC (SEQ ID NO: 127) TTN titin ENSCAFT000 chr36_25207752-26964E >  Substitution Nonsynonymous GTTTTNT 0.32 00022319 25207752_C_TK coding GCAT (SEQ ID NO: 128) TTN titin ENSCAFT000 chr36_25363681-6209E > K Substitution Nonsynonymous GTTCTNG 0.32 00022319 25363681_C_Tcoding TGAC (SEQ ID NO: 129) U5P45 ubiquitin  ENSCAFT000 chr12_60682412-232P > S Substitution Nonsynonymous GGGAGNA 0.43 specific 0000563860682412_G_A coding AAAA peptidase 45 (SEQ ID NO: 130) 04- OSA_(c) ASTN1astrotactin 1 ENSCAFT000 chr7_25651338- 762A > V SubstitutionNonsynonymous TGTGGNC 0.26 R04 00022524 25651338_C_T coding TTGT (SEQ IDNO: 131) ASXL3 additional sex ENSCAFT000 chr7_59080331- 1100P > LSubstitution Nonsynonymous CGGCCN 0.33 combs like 3 0002855159080331_G_A coding GAGGC (Drosophila) (SEQ ID NO: 132) FRMPD4FERM and PDZ ENSCAFT000 chrX_9178376- 1180A > T SubstitutionNonsynonymous TGGACNC 0.17 domain 00018460 9178376_G_A coding GGGCcontaining 4 (SEQ ID NO: 133) MC4R melanocortin ENSCAFT000chr1_19140979- 47V > I Substitution Nonsynonymous TCTTCNT 0.33receptor 4 00000145 19140979_G_A coding CTCC (SEQ ID NO: 134) MGAMmaltase- ENSCAFT000 chr16_10143723- NA Deletion Frameshift GGGTGNT 0.24glucoamylase 00006194 10143723_T_ TTTT (SEQ (alpha- ID NO: glucosidase)135) NFATC1 nuclear factor  ENSCAFT000 chr1_4124943- 8V > A SubstitutionNonsynonymous AAAGGNC 0.4 of activated T- 00000013 4124943_A_G codingTGGA cells, (SEQ ID cytoplasmic, NO: 136) calcineurin- dependent 1NFE2L3 nuclear factor ENSCAFT000 chr14_42452261- NA Deletion FrameshiftAAGATNA 0.3 (erythroid- 00038557 42452264_GATG TGTA derived 2)- _(SEQ ID like 3 NO: 137) TP53 cellular tumor ENSCAFT000 chr5_35558664-260F > S Substitution Nonsynonymous CCTCANA 0.54 antigen p53 0002646535558664 A G _ _ coding GCTG (SEQ ID NO: 138) PLEKHB pleckstrinENSCAFT000 chr21_27601782- 142R > H Substitution Nonsynonymous CTCGGNG0.43 1 homology 00009009 27601782_C_T coding GCTC domain (SEQ IDcontaining, NO: 139) family B (evectins) member 1 PTPN14 protein ENSCAFT000 chr7_15317710- 911G > R Substitution Nonsynonymous CATTCNC0.12 tyrosine 00019934 15317710_C_T coding TCTT phosphatase, (SEQ IDnon-receptor NO: 140) type 14 RBBP6 retinoblastoma ENSCAFT000chr6_24499626- 1730K > R Substitution Nonsynonymous TCTTTNT 0.3binding protein 00027846 24499626_T_C coding GCTG 6 (SEQ ID NO: 141)TDRD6 tudor domain ENSCAFT000 chr12_17857549- 1517W >  SubstitutionNonsense AACTGNT 0.49 containing 6 00003223 17857549_G_A X ATAA (SEQ IDNO: 142) TEX15 testis  ENSCAFT000 chr16_36456696- 1265V > F SubstitutionNonsynonymous TTTCANTT 0.58 expressed 00010405 36456696_G_T codingTTG (SEQ 15 ID NO: 143) TRAP1 TNF receptor- ENSCAFT000 chr6_40616562-42A > D Substitution Nonsynonymous TCCAGNC 0.3 associated 0003058440616562_C_A coding CAGT protein 1 (SEQ ID NO: 144) 04- STS- KIAA121uncharacterized ENSCAFT000 chr2_11859851- 356A > V SubstitutionNonsynonymous GAGAGNC 0.45 R02 PNST 7 protein 00006799 11859851_G_Acoding GGGG (SEQ ID NO: 145) MFSD2B major  ENSCAFT000 chr17_21486565-494R > C Substitution Nonsynonymous GTGCANG 0.42 facilitator 0000634121486565_C_T coding TGGG superfamily (SEQ ID domain NO: 146)containing 2B Novel uncharacterized ENSCAFT000 chrX_123930541- 327R > PSubstitution Nonsynonymous AGGGCNC 0.14 gene protein 00030447123930541_G_C coding CCCG (SEQ ID NO: 147) SLC16A2 solute carrierENSCAFT000 chrX_60903455- 72A > T Substitution Nonsynonymous CCTTCNC 0.4family 16, 00027229 60903455_G_A coding CTTT member 2 (SEQ ID (thyroidNO: 148) hormone transporter) TEP1 telomerase- ENSCAFT000chr15_20729329- 1900L > F Substitution Nonsynonymous CAGGANG 0.42associated 00008693 20729329_G_A coding CCCC protein 1 (SEQ ID NO: 149)XPNPEP X-prolyl ENSCAFT000 chrX_104033303- 502R > X SubstitutionNonsense CAGGGN 0.25 2 aminopeptidase 00029688 104033303_C_T GAATG(aminopeptidas (SEQ ID e P) 2, NO: 150) membrane- bound 01- STS- ACDadrenocortical ENSCAFT000 chr5_84799806- 388P > H SubstitutionNonsynonymous TGGCCNC 0.13 R02 PNST dysplasia 00032411 84799806_C_Acoding CTGC homolog (SEQ ID (mouse) NO: 151) ADAMTS ADAM ENSCAFT000chr31_25306205- 226H > Y Substitution Nonsynonymous CTGATNC 0.13 5metallopeptidas 00013627 25306205_G_A coding TGCC e with (SEQ IDthrombospondin NO: 152) type 1 motif, 5 ADRB2 beta-2 ENSCAFT000chr4_63253706- 76C > Y Substitution Nonsynonymous CAGCANA 0.12adrenergic 00029135 63253706_C_T coding GGCC receptor (SEQ ID NO: 153)ATP7B copper- ENSCAFT000 chr22_3160667- 1119V >  SubstitutionNonsynonymous TGGGCNT 0.2 transporting 00006859 3160667_G_A M codingGGCC ATPase 2 (SEQ ID NO: 154) CDK14 cyclin- ENSCAFT000 chr14_19522937-102R > W Substitution Nonsynonymous TCAGGNG 0.2 dependent 0000300919522937_C_T coding GCAC kinase 14 (SEQ ID NO: 155) IER5Limmediate early ENSCAFT000 chr9_57855189- 205 > N SubstitutionNonsynonymous CCACANC 0.16 response 5-like 00031805 57855189_G_A codingTCCC (SEQ ID NO: 156) IRS1 insulin  ENSCAFT000 chr25_42687032- 139S > NSubstitution Nonsynonymous CCGAGNT 0.11 receptor 00016522 42687032_C_Tcoding GCCG substrate 1 (SEQ ID NO: 157) JAG1 jagged 1 ENSCAFT000chr24_14655994- 935 > N Substitution Nonsynonymous CTGTANC 0.11 0000907414655994_G_A coding TTCG (SEQ ID NO: 158) JUNB jun B proto- ENSCAFT000chr20_52362490- 775 > L Substitution Nonsynonymous GCTCCNA 0.14 oncogene00027182 52362490_G_A coding TGAG (SEQ ID NO: 159) LMNA lamin A/CENSCAFT000 chr7_44690367- 64T > I Substitution Nonsynonymous ACTCGNT0.15 00026695 44690367_G_A coding GATG (SEQ ID NO: 160) MADCA mucosalENSCAFT000 chr20_61126306- NA Deletion Frameshift AAAGTNG 0.27 M1addressin cell 00031356 61126306_G_ GGGG adhesion (SEQ ID molecule 1NO: 161) precursor MEFV Mediterranean ENSCAFT000 chr6_41024970- 673N > KSubstitution Nonsynonymous GGAAANA 0.26 fever 00037775 41024970_C_Acoding AGAC (SEQ ID NO: 162) Novel Aldehyde ENSCAFT000 chr18_52833141-250V > A Substitution Nonsynonymous ACAGGNC 0.11 Gene dehydrogenase00017771 52833141_A_G coding GTAG (SEQ ID NO: 163) NRM nurim (nuclearENSCAFT000 chr12_3488483- 524S > N Substitution Nonsynonymous GGCAGNT0.11 envelope 00000694 3488483_C_T coding GCGG membrane (SEQ ID protein)NO: 164) PIM1 proto-oncogene ENSCAFT000 chr12_9213964- 73G > DSubstitution Nonsynonymous CCCCGNC 0.22 serine/ 00002258 9213964_G_Acoding TCCT  threonine (SEQ ID -protein NO: 165) kinase pim-1 PIM1proto-oncogene ENSCAFT000 chr12_9214807- 250H > Y SubstitutionNonsynonymous ACTGCNA 0.22 serine/ 00002258 9214807_C_T coding CAAC threonine (SEQ ID -protein NO: 166) kinase pim-1 PIM1 proto-oncogeneENSCAFT000 chr12_9214750- 231Q > X Substitution Nonsense CCCTGNA 0.2serine/ 00002258 9214750_C_T GGAG  threonine (SEQ ID -protein NO: 167)kinase pim-1 PTCH1 Patched-like ENSCAFT000 chr1_74305255- 73A > TSubstitution Nonsynonymous GGAAANC 0.16 protein 1 00001978 74305255_G_Acoding TACT (SEQ ID NO: 168) TRPS1 trichorhino- ENSCAFT000chr13_18226051- 5305 > N Substitution Nonsynonymous CATGANT 0.13 phala00001274 18226051_C_T coding GTCC ngeal syndrome (SEQ ID I NO: 169)ZFP36L1 zinc finger ENSCAFT000 chr8_45703888- 145 > N SubstitutionNonsynonymous CTTCGNT 0.13 protein 36, C3H 00026141 45703888_C_T codingCAAG type-like 1 (SEQ ID NO: 170) STS - soft tissue sarcoma; STS-PNST -soft tissue sarcoma, peripheral nerve sheath tumor; OSA, -chondroblastic osteosarcoma.

TABLE 4a Types of somatic changes observed across canine soft tissuesarcomas Number of Percentage of Type Subtype alterations alterations(%) Substitutions Nonsense 11 6 Missense (non- 135 73 synonymous) Splicesite acceptor 1 1 Splice site donor 4 2 Subtotal 151 82 INDELs Deletion4 2 Insertion 1 1 Subtotal 5 3 CNAs Deletion 0 0 Amplification 28 15Subtotal 28 15 Total 184 100 INDELs—insertions and deletions; CNAs—copynumber alterations

TABLE 4b Type of somatic mutations across canine soft tissue sarcomasType of somatic alteration Number Percentage 1 bp deletion 3 1.9 3 bpdeletion 1 0.6 1 bp deletion 1 0.6 A:T > C:G 3 1.9 A:T > G:C 4 2.6 A:T >T:A 3 1.9 C:G > A:T 4 2.6 C:G > G:C 2 1.3 C:G > T:A 71 45.5 G:C > A:T 5334.0 G:C > C:G 3 1.9 G:C > T:A 4 2.6 T:A > A:T 1 0.6 T:A > C:G 1 0.6T:A > G:C 2 1.3 Total 156 100Amplifications and deletions were less common, with an average of threeper tumor (range: of 0-17) (FIG. 5). Seven of the 10 soft tissuesarcomas harbored no amplifications or deletions. The chondroblasticosteosarcoma exome was similar to those of the soft tissue sarcomas,with 14 somatic mutations and four amplifications (Table 3 and FIG. 6).

Single base substitutions were identified in four tumor suppressor genesthat are frequently mutated in human tumors (NF1, MLL3, TP53, andPTCH1). Additionally, MDM4, an oncogene that has been shown to beamplified but not point-mutated in human cancers was found to beamplified (but not point-mutated) in one canine tumor (Lee, et al.,2012, Barretina, et al., 2010, Chmielecki, et al., 2013, Vogelstein, etal., 2013). The only genes mutated in more than one tumor were ATP7B(missense mutations in two tumors) and A/G1 (amplified in two tumors).Interestingly, mutations in ATP7B were also found in a humanliposarcomas (Joseph, et al., 2013). Twenty-two of the 184 somaticmutations in canine tumors occurred in genes previously shown to bemutated in human soft tissue sarcomas (Table 5).

TABLE 5 Genes mutated in both human and canine cancers Human driverNumber Number gene or mutated of somatic Type of of in human soft Genealterations alteration samples tissue sarcoma ANKRD11 1 SBS 1 Joseph etal., (splice site) 2013 ATP7B 2 SBS 2 Joseph et al., (missense) 2013BRDT 1 SBS 1 Chemielecki (missense) et al., 2013 BRWD3 1 SBS 1 Joseph etal., (missense) 2013 CSMD2 1 SBS 1 Joseph et al., (missense) 2013 FCRLB1 SBS 1 Lee et al., (missense) 2012 IRS1 1 SBS 1 Barretina (missense) etal., 2010 LIMK1 1 SBS 1 Lee et al., (missense) 2012 MBD5 1 SBS 1 Lee etal., (missense) 2012 MLL3 1 Deletion 1 Vogelstein et al., 2013 NF1 1 SBS1 Barretina (missense) et al., 2010 PKHD1 1 SBS 1 Lee et al., (missense)2012 PTCH1 1 SBS 1 Vogelstein (missense) et al., 2013 PTPRZ1 1 SBS 1Chemielecki (missense) et al., 2013 RP1 1 SBS 1 Chemielecki (missense)et al., 2013 TTN 4 SBS 1 Chemielecki (missense) et al., 2013 MDM4 1Amplification 1 Vogelstein et al., 2013 CNTN2 1 Amplification 1Chemielecki et al., 2013Larger studies of soft tissue sarcomas in both species will be requiredto determine whether these represent driver mutations that signifyimportant, conserved tumorigenic pathways. Regardless, the geneticlandscapes of canine tumors were similar to those of humans in terms ofthe numbers of genetic alterations and spectrum of mutations.Specifically, they exclude the possibility that the canine tumors have avery large number of mutations which might make them more likely tomount an immune response than analogous tumor types in humans.

Example 6 Intratumoral (IT) Administration of C. novyi NT—Study 1Methods

To investigate the safety and efficacy of the method of the presentinvention, a comparative study in 16 dogs with spontaneously occurringsolid tumors was performed (Table 6).

TABLE 6 Patient Characteristics Body Longest # of IT Age Weight Tumordiameter^(d) Previous C. novyi-NT Case ID Sex^(a) Breed (years) (kg)type^(b) Grade^(c) Location (mm) treatment treatments 01-R02 FN Border14.3 21.7 STS- II Left flank 43 None 4 collie PNST 04-R01 MN Golden 7.934.0 STS- II Right maxilla 15 Surgical 4 retriever PNST 04-R02 Ml Golden12.0 38.8 STS- I Right lateral 46 Surgical 4 retriever PNST metacarpus04-R03 MN Boxer 9.6 29.4 STS I Left medial 56 None    3^(TR)antebrachium 04-R04 FN St. 11.7 31.0 OSA_(c) III Right proximal NDSurgical  1^(AE) Bernard humerus 04-R05 MN Shetland 14.0 13.4 STS IIIRight cranial 45 Surgical & 4 sheepdog antebrachium C. novyi- NT sporesIV 04-R06 FN Labrador 11.6 24.3 MCT III Right hindlimb 23 None 4retriever digit III 04-R08 FN Shepherd 7.2 28.9 STS- I Right medial 65Surgical    3^(PD) PNST hindlimb paw 10-R01 MN Golden 13.7 33.6 OMM IIILeft mandible 27 Surgical  2^(AE) retriever 10-R02 MN Pit bull 10.0 43.6STS I Right flank 53 Surgical 4 terrier 11-R01 MN Maltese 11.1 8.1 STS-II Left pinna 28 Surgical    1^(TR) PNST 11-R02 FN Labrador 12.2 30.3STS- II Left stifle 43 None  3^(IV) retriever PNST 11-R04 MN Husky 10.344.3 STS I Right forelimb 29 None 4 paw 16-R02 MN Labrador 9.8 36.8 STSI Left lateral 91 Surgical 4 retriever thigh 16-R03 FN Shepherd 10.820.8 STS I Left forelimb 53 Surgical 4 paw 26-R01 MN Labrador 7.9 30.8STS II Right forelimb 24 None 4 retriever paw ^(a)FN—female neutered;MN—male neutered; MI—male intact. ^(b)STS—soft tissue sarcoma; STS -PNST—peripheral nerve sheath tumor; OSA_(c)—chondroblastic osteosarcoma;MCT—mast cell tumor; OMM—oral malignant melanoma. ^(c)Grading based onpublished criteria (Dennis et al., 2011, Patnaik et al., 1984, Smedleyet al., 2011, Sabattini et al., 2014): I—low grade; II—intermediategrade; III—high grade; NA—not assessed. ^(d)Longest diameter at time offirst C. novyi -NT administration (day 0). ND—unmeasurable due tolocation. ^(e)04-R05—previous C. novyi -NT therapy with a single IVinjection of 1 × 10⁷ spores/m² 437 days prior to the first ITadministration of C. novyi-NT spores. ^(f)Reason for number oftreatments less than 4 given in superscript: TR—tumor response;AE—adverse event; PD—progressive disease; IV—4th dose givenintravenously.

Dogs were enrolled at multiple sites participating in the AnimalClinical Investigation oncology network (ACI, Washington, D.C.) andwritten informed consent was obtained from owner(s) prior to enrollment.Treatment, management, and study evaluations were overseen byboard-certified veterinary oncologists. Enrollment was offered toclient-owned dogs with spontaneous solid tumors, with a preference forsoft-tissue sarcomas that had failed standard therapy or whose owner(s)had declined such therapy. Participation was restricted to tumor bearingdogs with a target lesion having a longest diameter between 1 and 7centimeters. Dogs with tumors located in areas where abscess developmentwould be catastrophic (e.g., nasal tumors that extended into the brainor significant pulmonary metastatic disease) were excluded from thestudy.

Dogs with evidence of an active bacterial infection requiring systemicantibiotic therapy within seven days or cancer therapy (chemotherapy,radiation therapy, and immunotherapy) within 21 days of C. novyi-NTspore treatment were ineligible. Dogs were required to have aperformance score of 0 or 1 (Table 7) and to be available for the fullduration of the study for enrollment. Concurrent use of anticanceragents and participation in other clinical trials were prohibited. Dogsthat were pregnant or likely to become pregnant were not included in thestudy. Also, dogs that may have been unavailable for the entire studyduration, and dogs that were considered unsuitable for study enrollmentby the Investigator or Medical Director were not included in the study.

TABLE 7 Performance status evaluations Score Description 0 Normalactivity 1 Restricted activity: decreased activity from pre-diseasestatus 2 Compromised: ambulatory only for vital activities, able toconsistently defecate and urinate in acceptable areas 3 Disabled: mustbe force fed and/or unable to confine urination and defecation toacceptable areas 4 Death

During a screening visit, each dog was assigned a unique study dogidentification number consisting of a 5-digit numeric code (which maynot have been sequentially in order of the screening dog number). Thefirst 2 digits indicated the study site (01 to 99), the middle digitindicated the study ‘R’, and the last 2 digits described the study dognumber within a study site (01 to 99). For example the 11th dog enrolledat Site 9 was assigned study dog number 09-R11. Study dog numbers wereassigned chronologically in the order that dogs were enrolled at a givenstudy site. A dog was considered enrolled in the study when it satisfiedthe inclusion and exclusion criteria.

Gross pathology and histopathology was performed in accordance with Foodand Drug Administration's CVM Guidance for Industry 185. At necropsy,the following tissues (Table 8) were assessed for gross pathology andfor histopathology and described in the necropsy report. Samples ofbrain, heart, lung, liver, spleen, kidney, muscle, bone, smallintestine, large intestine and any tissue with gross abnormality werecollected for microbiology.

TABLE 8 List of tissues to be examined by gross pathology andhistopathology Pituitary gland Thyroid gland Parathyroid gland Adrenalgland Pancreas Ovaries Uterus Testes Prostate Epididymis HeartVentricles Brain Spinal cord Eyes Lung Muscle Mammary gland Liver Gallbladder Kidneys Urinary bladder Lymph nodes Skin Bone and marrow Marrowsmear Spleen Stomach Duodenum Jejunum Ileum Colon Cecum Thymus Injectionsite Any abnormal tissues

All dogs were hospitalized from day 0 (DO) to day 4 (D4), and thenoptionally (at the Investigator's discretion) for 24 to 48 hours aftereach subsequent treatment for clinical observation. Fluids wereadministered to all study dogs during hospitalization following C. novyiNT treatment. On dosing days all dogs were administered intravenous (IV)crystalloids at 4 ml/kg/h for 2 hours. Dogs were closely monitored forsix hours after each IT injection of C. novyi-NT spores. At the nextvisit (4 days later) all dogs were administered subcutaneous (SQ)crystalloids at 20 ml/kg. If a dog was hospitalized and receiving IVcrystalloids on the day that SQ crystalloids were to be administered, itwas not necessary to give the SQ dose.

Study visits and events are summarized in Table 9 as an example of a4-dose treatment regimen. The dosing interval was suggested to be on aweekly basis, if the dog was to be treated with repeated dosing.Treatment delays for repeated dosing occurred during the course of thestudy due to adverse events or the decision of the investigator.

TABLE 9 Summary of study evaluations Pretreatment Screening^(a) Day0^(b) Day 4 Day 7^(b) Day 11 Day 14^(b) Day 18 Day 21^(b) Day 25 Day 60Day 90 Informed X Consent Medical X History & Demographics Physical ExamX X X X X X X X X X X Weight & Vital X X X X X X X X X X X SignsPerformance X Score Inclusion & X Exclusion Criteria Laboratory X X X X(X) (X) (X) (X) (X) (X) (X) Values^(c) Imaging^(d) X (X) (X) (X) (X) (X)(X) (X) (X) (X) (X) Biopsy X Research X Bloodwork Tumor X X X X X X XMeasurements & Photographs Assign study X dog number Enrollment X IT C.novyi-NT X X X X X IV Fluid X X X X X Therapy^(e) SQ Fluid X X X XTherapy^(f) Study X completion^(g) ^(a)Screening evaluations undertaken1-14 days prior to treatment. ^(b)Patient monitored 6 hourspost-treatment. Evaluation made every 15 minutes for 1st hourpost-treatment, every 30 minutes for 2nd hour post treatment and every60 minutes for 3rd-6th hour post-treatment. ^(c)Laboratory valuesinclude: complete blood count, serum biochemistry panel, prothrombintime, thromboplastin time and urinalysis. (X)—at discretion of theinvestigator. ^(d)Diagnostic imaging including: radiographs, ultrasoundexamination, or computed tomography. ^(e)Crystalloid at 4 mL/kg/hr fortwo hours. ^(f)Crystalloid at 20 mL/kg. ^(g)Following study completionand if systemic antibiotics were required to manage adverse events, itwas recommended to administer doxycycline 5-10 mg/kg orally twice a day(PO BID) to dogs for 3 months.

Sixteen dogs, 9 neutered males, 1 entire (intact) male, and 6 neuteredfemales, were enrolled in the study. (Table 6). Their demographics andtumor characteristics are given in Table 6. Enrolled cases exhibiteddiverse breeds, weights and ages. Cases were previously diagnosed withnaturally occurring cancers representing a variety of histologicalorigins: 13 dogs had a diagnosis of soft tissue sarcoma (81.3%), 1osteosarcoma (6.3%), 1 melanoma (6.3%) and 1 mast cell tumor (6.3%). Ofthe 13 soft tissue sarcomas, histologic subtype was available for 11 andincluded: 4 hemangiopericytomas (30.8%), 3 peripheral nerve sheathtumors (23.1%), 1 synovial cell sarcoma (7.7%), 1 myxosarcoma (7.7%), 1rhabdosarcoma (7.7%) and 1 fibrosarcoma (7.7%). The mean weight of dogsin the trial was 29.4 kg (range 8.1-44.3 kg) and their mean age was 10.9years (range: 7.2-14.3 years). Thirteen dogs had a diagnosis of softtissue sarcoma, and one each had a diagnosis of osteosarcoma, malignantmelanoma, and mast cell tumor. Of the 13 soft tissue sarcomas, six wereavailable for immunohistochemistry (IHC). All six were positive for S100and negative for smooth muscle actin, suggesting the diagnosis of asarcoma subtype called peripheral nerve sheath tumors. Seven of thetumors were grade I, five were grade II, and four were grade III. Eightdogs had previous surgical therapy for their cancers.

Preparation and IT Injection of C. novyi-NT Spores in Spontaneous CanineTumors

C. novyi-NT spores for use in the comparative canine study were producedas previously described (Dang, et al., 2001, Bettegowda, et al., 2006).In brief, bacteria were cultured in sporulation medium for at least twoweeks to ensure maximum yield of mature spores. Mature spores werepurified through two consecutive, continuous Percoll gradients followedby four washes and re-suspensions in PBS. Sterility testing of the finalproduct was performed by culturing product in Soybean-Casein DigestMedium and Thioglycollate Medium in accordance with FDA 21CFR610.12guidelines (Nelson Laboratories, Salt Lake City, Utah). Germinationefficiency assays were performed under anaerobic conditions on Brucellaagar with 5% horse blood to ensure the spores meet preset viabilitycriteria. Spores were packaged in sterile 1.8 mL cryovials with O-ringsealed screw caps (Simport, Beloeil, Canada) at a volume of 1000 μL anda concentration of 1×10⁹ spores/mL. C. novyi-NT cryovials were stored at2-8° C. For dosing, a 0.4 mL aliquot of the stock spore solution waspackaged into 0.5 mL cryovials. After dosing, the cryovials and unusedC. novyi-NT spores were discarded according to applicable regulationsfor disposal of Biosafety Level 2 material. Prior to IT injection,spores were re-suspended with a vortex, mixing at maximum speed for 10seconds for a total of three times before being withdrawn into a 1 mLsyringe. The injection site was aseptically prepared. If available,ultrasound or computed tomography (CT) was used to identify a necroticregion of the tumor. If a necrotic region was not identified, theinjection was directed to the center of the tumor. The needle wasinserted once into the pre-defined region and 100 μL of spore suspension(1×10⁸ C. novyi-NT spores) were dispensed with even pressure. Theinjection needle was removed slowly and the injection site sterilized.All dogs received at least 1 cycle of an IT dose of 1×10⁸ spores in 100μL saline (biosurgery): 3 dogs received a single treatment cycle, 13dogs received more than 1 and up to 4 treatment cycles. Dogs couldreceive up to 4 cycles of biosurgery with a one-week interval betweencycles. Treated dogs were followed for at least 90 days after the firstIT injection. Extended follow-up for disease progression and survivalwere warranted when available. Early withdrawal from the study wasallowed for toxicity or progressive disease.

Study evaluations were undertaken as described in Table 9. Pre-screeningevaluations were conducted 1 to 14 days before the first cycle ofbiosurgery. Dogs were monitored periodically on both an inpatient andoutpatient basis during the study. Laboratory samples were taken asdefined in Table 9 and included a complete blood count, serumbiochemistry, prothrombin time, partial thromboplastin time, andurinalysis. Imaging was performed at screening and included regional CT,thoracic radiography, and abdominal ultrasonography. Additional imagingmay be conducted during the study at the investigator's discretion.

Adverse events were evaluated, where possible, using the VeterinaryCo-operative Oncology Group—Common Terminology Criteria for AdverseEvents (VCOG-CTCAE) v1.0 (Veterinary Co-operative Oncology Group, 2004),with terminology from the Veterinary Dictionary for Drug Related Affairs(VeDDRA) rev.4 (European Medicines Agency, 2012). Terminologies foradverse events related to C. novyi-NT germination (target lesionreactions) are defined in Table 10. Clinical observations withoutappropriate VeDDRA or target lesion reaction terminology were classifiedseparately as uncoded signs (Table 11). Relationship to C. novyi-NTtherapy was determined by the reporting investigator.

TABLE 10 Coded terms to describe tumor adverse events associated with C.novyi-NT activity System Organ Class High Level Preferred Low Level(SOC) Term Term (HLT) Term (PT) Term (LLT) Target lesion Tumor Tumorabscess Tumor abscess reaction inflammation Target lesion Tumor Tumorabscess Tumor closed reaction inflammation wound Target lesion TumorTumor abscess Tumor reaction inflammation malodorous Target lesion TumorTumor abscess Tumor necrosis reaction inflammation Target lesion TumorTumor abscess Tumor open reaction inflammation wound Target lesion TumorTumor abscess Tumor tissue loss reaction inflammation Target lesionTumor Tumor abscess Tumor tissue reaction inflammation sloughing Targetlesion Tumor Tumor abscess Tumor ulceration reaction inflammation Targetlesion Tumor Tumor Tumor reaction inflammation consistency consistencychange change Target lesion Tumor Tumor Tumor firmer reactioninflammation consistency change Target lesion Tumor Tumor Tumor softerreaction inflammation consistency change Target lesion Tumor Tumordischarge Tumor bleeding reaction inflammation Target lesion Tumor Tumordischarge Tumor bloody reaction inflammation discharge Target lesionTumor Tumor discharge Tumor discharge reaction inflammation Targetlesion Tumor Tumor discharge Tumor purulent reaction inflammationdischarge Target lesion Tumor Tumor discharge Tumor serous reactioninflammation discharge Target lesion Tumor Tumor Increased tumorreaction inflammation inflammation heat Target lesion Tumor TumorIncreased tumor reaction inflammation inflammation warmth Target lesionTumor Tumor Tumor reaction inflammation inflammation edematous Targetlesion Tumor Tumor Tumor reaction inflammation inflammation inflammationTarget lesion Tumor Tumor Tumor reaction inflammation inflammationinflammatory reaction Target lesion Tumor Tumor Tumor pruritis reactioninflammation inflammation Target lesion Tumor Tumor Tumor swollenreaction inflammation inflammation Target lesion Tumor Tumor pain Tumorpain reaction inflammation Target lesion Tumor Tumor skin Tumor bruisingreaction inflammation disorder Target lesion Tumor Tumor skin Tumorreaction inflammation disorder discoloration Target lesion Tumor Tumorskin Tumor erythema reaction inflammation disorder Target lesion TumorTumor skin Tumor reaction inflammation disorder petichiation Targetlesion Tumor Other tumor Other tumor reaction inflammation disorderdisorder Target lesion Tumor Tumor pain Tumor discomfort reactioninflammation

TABLE 11 Signs not attributable in VeDDRA to underlying clinical entityor C. novyi-NT related target lesion reaction # of dogs (with at AdverseEvent least 1 (Preferred occurrence Term) G-I G-II G-III G-IV of AE)Total Uncoded sign 15 2 1^(a) 5 18 ^(a)Grade IV decrease in bloodeosinophils reported by investigator.

Longest diameter tumor measurements of the target (injected) lesion weremade on day 0, day 7, day 14, day 21, day 60 and day 90 post-treatment(Table 9). Non-target and new lesions were recorded but not measured.The best overall target response was evaluated on or after the day 21study visit: complete response (CR) was defined as the completedisappearance of the target lesion; partial response (PR) was defined asat least a 30% decrease in the longest diameter of the target lesion;and progressive target disease (PD) was defined as at least a 20%increase in the longest diameter of the target lesion or the appearanceof new nontarget lesions. Stable disease (SD) was defined asinsufficient decrease or increase in the longest diameter of the targetlesion to qualify as CR, PR, or PD. In the case of C. novyi-NT relatedabscesses, medical, or surgical debridement of necrotic tissue was atthe discretion of the investigator.

Evaluation of surgical samples and necropsies were conducted by boardcertified veterinary pathologists. Tissue specimens were fixed in 10%neutral buffered formalin and embedded in paraffin. Slides stained withH&E and or gram stained slides were prepared for evaluation according tostandard procedure guidelines. For immunohistochemistry (IHC),formalin-fixed, paraffin-embedded tumor tissue was sectioned at 5 μm,deparaffinized in xylene, and rehydrated through graded alcohols.Antigen retrieval was done using unmasking solution (VectorLaboratories, Burlingame, Calif.). Primary antibodies S100 (DAKO,Carpinteria, Calif.) and anti-smooth muscle actin (DAKO, Carpinteria,Calif.) were used at 1:100. Secondary antibodies (Vector Laboratories,Burlingame, Calif.) labeled with DAB were used at 1:500. Sections wereincubated with ABC reagent (Vector Laboratories, Burlingame, Calif.) andcounterstained with hematoxylin. Tumor grades were assigned to eachbased on published criteria (Dennis, et al., 2011, Patnaik, et al.,1984, Smedley, et al., 2011, Sabattini, et al., 2014).

Example 7 Intratumoral (IT) Administration of C. novyi-NT—Study 1Results

All dogs received at least one cycle of biosurgery, with 53 cycles givenof a maximum of 64 planned. The majority of dogs, 10 of 16, received theintended four cycles. Cycles of biosurgery were typically one weekapart. No placebo control or masking was used.

For dogs showing early tumor responses, toxicity, or progressive diseaseafter the first cycle, subsequent cycles were stopped. The most commonadverse events were consistent with local infection at the C. novyi-NTspore injection site, including: fever (17 incidents), tumorinflammation (12 incidents), tumor abscess (10 incidents), anorexia(nine incidents), and lethargy (six incidents) (Table 12). Clinicalsigns of an inflammatory response at the injected target lesion site wasobserved in 14 of 16 dogs (87.5%), including: tumor inflammation(12/14), tumor abscess (7/14), tumor pain (5/14), and tumor discharge(4/14) (Table 13).

TABLE 12 Summary of adverse events observed throughout study # of dogs(with at least 1 Adverse Event occurrence (Preferred Term) G-I G-IIG-III G-IV of AE) Total Hyperthermia 14 3 10 17 Tumor inflammation 7 4 112 12 Tumor abscess 6 3 1 8 10 Anorexia 7 2 8 9 Lethargy 3 2 1 6 6Lameness 5 1 6 6 Oedema 5 1 5 6 Hypertension 6 4 6 Neutrophilia 6 6 6Tumor discharge 6 4 6 Anaemia 4 1 5 5 Diarrhoea 3 1 2 4 Tumor pain 3 1 44 Leucocytosis 4 3 4 Lymphadenitis 4 4 4 Tumor consistency 3 3 3 changeLeucopenia 1 1 1 2 Thrombocytopenia 1 1 2 2 Localized pain 1 1 2 2Lymphopenia 1 1 2 2 Change in blood protein 1 1 2 2 Emesis 1 1 2 2 Fluidin abdomen 1 1 1 2 General pain 1 1 2 2 Electrolyte disorder 2 2 2Impaired consciousness 2 2 2 Tumor skin disorder 2 2 2 Neutropenia 1 1 1Malaise 1 1 1 Muscle weakness 1 1 1 Recumbency 1 1 1 Steatitis 1 1 1Digestive tract 1 1 1 haemorrhage Skin and tissue 1 1 1 infectionArrhythmia 1 1 1 Bone and joint disorder 1 1 1 Cardiac enlargement 1 1 1Digestive tract disorder 1 1 1 Eosinophilia 1 1 1 Erythema 1 1 1Hepatomegaly 1 1 1 Hepatopathy 1 1 1 Injection site pruritus 1 1 1Lymphocytosis 1 1 1 Murmur 1 1 1 Nausea 1 1 1 Palpable mass 1 1 1Pulmonary disorder 1 1 1 Skin haemorrhage 1 1 1 Urine abnormalities 1 11 Total 153

TABLE 13 Summary of clinical evidence of germination and response fromC. novyi-NT therapy Clinical Case ID Clinical evidence ofgermination^(a) response^(b) 01-R02 Tumor inflammation, skin disorderand PD discharge 04-R01 Tumor inflammation and pain CR 04-R02 Tumorinflammation and abscess PR 04-R03 Tumor inflammation, consistencychange, CR discharge and tumor pain 04-R04 Tumor inflammation and painNE 04-R05 Tumor inflammation, consistency change, PR skin disorder andpain 04-R06 Tumor inflammation, abscess and discharge CR 04-R08 Tumorabscess and discharge NE 10-R01 — PD 10-R02 Tumor inflammation, abscessand pain SD 11-R01 Tumor inflammation and abscess PR 11-R02 Tumorinflammation SD 11-R04 Tumor abscess and consistency change SD 16-R02Tumor inflammation PD 16-R03 Tumor inflammation and abscess SD 26-R01 —SD ^(a)Clinical evidence of C. novyi-NT germination on or after day 0 ofthe study and includes target lesion reactions (FIG. 5). ^(b)Bestresponse of the target lesion, as defined by the study protocol, afterday 21 of the study: CR—complete response; PR—partial response;SD—stable disease; PD—progressive disease; NE—not evaluable for responseafter on or after day 21 of the study.

Early-Onset Adverse Events

Early-onset adverse events refer to the events occurring within thefirst 7 days following the first treatment cycle (13 dogs) or a singletreatment cycle (3 dogs). A variety of adverse (AE) event findings werenoted across multiple cases. The early-onset adverse events thatoccurred within 7 days either after the 1^(st) treatment cycle (13 dogsthat have received multiple cycles) or after the single treatment cycle(3 dogs that have received only one cycle) are summarized in Table 14.

TABLE 14 Summary of early onset^(a) adverse events of any grade duringthe first treatment cycle Number Adverse of dogs^(b) Incidence EventType (N = 16) (%) Tumor Target Lesion reaction 9 56.3% inflammationAnorexia General signs or symptoms 4 25.0% Edema General signs orsymptoms 4 25.0% Fever General signs or symptoms 4 25.0% WBC increasedBlood and lymphatic system 2 12.5% Hypertension Circulatory disorders 212.5% Lethargy General signs or symptoms 2 12.5% Pain General signs orsymptoms 2 12.5% Tumor abscess Target Lesion reaction 2 12.5% Hbdecreased Blood and lymphatic system 1 6.3% MCV decreased Blood andlymphatic system 1 6.3% Neutrophils Blood and lymphatic system 1 6.3%increased RBC decreased Blood and lymphatic system 1 6.3% WBC decreasedBlood and lymphatic system 1 6.3% Blood in feces Digestive tractdisorders 1 6.3% Diarrhea Digestive tract disorders 1 6.3% NauseaDigestive tract disorders 1 6.3% Regurgitation Digestive tract disorders1 6.3% Vomiting Digestive tract disorders 1 6.3% Injection siteInjection site reactions 1 6.3% pruritus Tumor bleeding Target Lesionreaction 1 6.3% Tumor erythema Target Lesion reaction 1 6.3% ^(a)Up toand less than 7 days after first treatment. ^(b)Number of dogs with atleast one adverse event of any grade

Common early onset adverse event findings included: target tumor lesionreactions, alterations in general signs and symptoms, and blood andlymphatic system abnormalities. The majority of early onset adverseevents were mild to moderate (Grade I-II), with tumor inflammation,anorexia, tumor edema, and fever being the most commonly observedevents. Grade III tumor abscess and Grade III tumor inflammation werenoted in two cases (10-R02 and 16-R03). Early onset adverse eventfindings appear consistent with the anticipated tumor inflammatoryreactions resulting from the mechanism of action of the C. novyi-NTtherapeutic.

Late-Onset Adverse Events

A subset of 3 dogs received only a single treatment cycle (as of Dec. 2,2012). Late-onset adverse events refer to the events occurring after 7days following the single treatment cycle and are summarized in Table 15for the 3 dogs (04-R4, 10-R02, and 11-R01). The majority of late-onsetadverse events were mild to moderate (Grade I-I) and 11 of the 12 lateronset findings were noted in a single subject 04-R04. This dog presentedwith chondroblastic osteosarcoma of the right forelimb with a LDmeasurement of 94.5 mm at baseline (CT measurement not available).Amputation was pursued 20 days after C. novyi-NT spore injection due toprogressive disease. The other two subjects have well tolerated thesingle treatment cycle. Their late-onset AE was exclusively limited to amild fever (Grade I).

TABLE 15 Summary of later onset^(a) adverse events of any grade afterfirst treatment cycle Number Adverse of dogs^(b) Incidence Days to EventType (N = 3) (%) Finding^(c) Fever General signs or symptoms 1 33.3% 9Pain General signs or symptoms 1 33.3% 20 Surgical site disorderSystemic disorders NOS 1 33.3% 24 Neutrophils increased Blood andlymphatic system 1 33.3% 34 RBC decreased Blood and lymphatic system 133.3% 34 Eosinophils increased Blood and lymphatic system 1 33.3% 61 WBCincreased Blood and lymphatic system 1 33.3% 61 Tumor new mass Neoplasia1 33.3% 82 Lymphadenopathy Lymph node disorders 1 33.3% 82 Thrombocytesdecreased Blood and lymphatic system 1 33.3% 93 ^(a)After 7 daysfollowing a single treatment only. ^(b)Number of dogs with at least oneadverse event of any grade. ^(c)From day of first treatment.

In summary, the safety profile observed following one treatment cycle ofC. novyi-NT IT administration of 1×10⁸ spores suggested suitabletolerability. The early-onset and late-onset adverse events wereconsistent with the anticipated tumor inflammatory reactions resultingfrom the mechanism of action of C. novyi-NT. The adverse events havebeen monitored and managed effectively as disclosed herein.

The adverse events noted when dogs were given multiple treatment cyclesof C. novyi-NT by IT administration are summarized in Table 9 foradverse events (AEs) of any Grades and in Table 10 for AEs of Grade IIIand above.

The variety and incidence of adverse event findings following multiplecycles of treatment was broadly similar to that observed following asingle treatment cycle. Likewise, the onset of events appeared to belargely consistent with what was observed following a single treatmentcycle: of 169 findings across all cases, only 30 were noted more thanseven days following a prior dose. Similarly, tumor inflammation,anorexia, and fever were the most commonly observed events. Adverseevents that occurred in more than one case included: target lesionreactions, alterations in general signs and symptoms, blood andlymphatic system abnormalities, lameness, hypertension, lymphadenopathy,diarrhea, and new masses. The majority (about 95%) of findings were mildto moderate in intensity (Grade I to II).

Severe Adverse Events

Severe adverse events (Grade III and greater) were noted in 5 cases(Table 16). Subject 04-R05 experienced a Grade III increase inneutrophil count. Subject 10-R01 experienced Grade III anemia, lethargy,muscle weakness, myositis, pain and recumbency. Extensive metastaticdisease, while not observed at baseline, was diagnosed followingnecropsy of case 10-R01 at Day 60; progressive disease may haveinfluenced adverse event findings in this case. Subject 10-R02experienced a Grade III tumor abscess. Subject 11-R01 experienced aGrade IV decreased thrombocyte count 93 days after first treatment cyclewhich resolved without intervention. Symptoms resolved 21 days after theDay 93 visit without any medical treatment. Notably, this subject alsoexhibited Grade I and Grade III symptoms of thrombocytopenia atscreening and baseline, respectively. Subject 16-R03 experienced GradeIII diarrhea, lameness and tumor inflammation that resolved within oneweek.

TABLE 16 Summary of adverse events greater than or equal to Grade IIIfor all treatment cycles Number Adverse of dogs^(a) Incidence Event Type(N = 16) (%) Lameness Musculoskeletal disorders 3 18.8% Pain Generalsigns or symptoms 2 12.5% Anemia Blood and lymphatic system 1 6.3%Neutrophils decreased Blood and lymphatic system 1 6.3% Thombocytesdecreased Blood and lymphatic system 1 6.3% Diarrhea Digestive tractdisorders 1 6.3% Lethargy General signs or symptoms 1 6.3% SteatitisGeneral signs or symptoms 1 6.3% Myositis Musculoskeletal disorders 16.3% Tumor abscess Target Lesion reaction 1 6.3% Tumor inflammationTarget Lesion reaction 1 6.3% ^(a)Number of dogs with at least oneadverse event of any grade.

Two dogs had documented new masses during the study. A rectal mass wasidentified in subject 04-R04 on Day 82 and a lytic vertebral lesion ofT1 in subject 10-R01 on Day 9. These findings may represent a metastasisor a second distinct pathology. In both cases, the relationship to C.novyi-NT therapy was unclear.

Response from C. novyi-NT Therapy

In summary, C. novyi-NT IT treatment in companion dogs at a dose of1×10⁸ spores per cycle of therapy for up to 4 cycles is well tolerated.Most adverse events possibly or probably related to drug that weregreater than Grade III resolved within one week. Expected adverse eventshave been largely associated with local inflammatory changes followingintratumoral therapy and generally resolved within one week. The adverseevents and serious adverse events have been monitored and managedeffectively as disclosed herein.

Given that C. novyi-NT IT administration was accompanied by broadevidence of biological activity, a preliminary assessment of primarytumor response using RECIST 1.1 was made and is summarized in Table 17below.

TABLE 17 Summary of clinical evidence of germination and response fromC. novyi-NT therapy Clinical evidence of Clinical Case IDgermination^(a) Response^(b) 01-R02 Tumor inflammation, PD skin disorderand disorder 04-R01 Tumor inflammation and CR pain 04-R02 Tumorinflammation and PR abscess 04-R03 Tumor inflammation, CR consistencychange, discharge and tumor pain 04-R04 Tumor inflammation and NE pain04-R05 Tumor inflammation, PR consistency change, skin disorder and pain04-R06 Tumor inflammation, CR abscess and discharge 04-R08 Tumor abscessand NE discharge 10-R01 — PD 10-R02 Tumor inflammation, SD abscess andpain 11-R01 Tumor inflammation and PR abscess 11-R02 Tumor inflammationSD 11-R04 Tumor abscess and SD consistency change 16-R02 Tumorinflammation PD 16-R03 Tumor inflammation and SD abscess 26-R01 — SD

Dogs were evaluated for best response on or after day 21 of the study.Three had a complete response (CR) to therapy, three had partialresponses (PR), five had stable disease (SD), three had progressivedisease (PD), and two dogs (04-R04 and 04-R08) were not evaluable forresponse because the injected tumor was surgically resected before day21. The objective response rate for biosurgery was 37.5% (6 of 16 dogs;95 percent confidence interval: 15.2-64.6%). Tumor abscesses andresponses occurred after one to four cycles of biosurgery. Dog 11-R01experienced a PR after a single cycle, 04-R03 had a CR after threecycles, dogs 04-R02 and 04-R05 had PRs after four cycles, while 04-R01and 04-R06 had CRs after four cycles. FIGS. 7A-F and FIGS. 8A-F showrepresentative changes in dogs with partial (11-R01) and completeresponses (04-R03), respectively. Resolution of abscesses occurred withdebridement and wound healing was complete after 2 to 4 weeks. However,overt abscess formation was not always observed before an objectiveresponse. Dogs 04-R01 and 04-R06 received 4 cycles of biosurgery, withtumor inflammation, but not abscessation, observed up to the day 21study visit. Even so, complete responses were noted on the day 42(unscheduled visit) and day 60 study visits in these two dogs,respectively.

Individual subjects are discussed in more detail below:

Andy (11-R01, FIGS. 7A-F), a 10 year-old, neutered male, Maltese,presented with a grade II soft tissue sarcoma on the left pinna. Histreatment history included surgery prior to enrollment. He received asingle dose of C. novyi-NT spores on Jun. 18, 2012. Andy experiencedGrade I tumor swelling on Day 1 (Jun. 19, 2012). Abscess formation ledto ulceration of the tumor and discharge of purulent, necrotic material.The resulting wound healed without complication. During the extendedfollow-up period, a Grade IV thrombocytopenia was observed on Day 93(Sep. 19, 2012) that resolved at a routine follow-up visit a few weekslater. A thickened cutaneous area of approximately 8 mm remained afterwound healing (see FIG. 9 for a time course of tumor measurements overthe course of the study). This may have represented scar tissue orresidual tumor.

Molly (11-R02), a 12 year-old, neutered female, Labrador Retriever,presented with a grade II soft tissue sarcoma on the left stifle. Shehad no treatment history prior to enrollment. She received 3 cycles ofIT C. novyi-NT spores, followed by 1 IV dose of 1×10⁸ C. novyi-NTspores, 7 days after the 3rd IT dose. Her 1st, 2nd and 3rd IT doses onJul. 11, 2012, Jul. 18, 2012, and Jul. 25, 2012, respectively. Thesingle IV dose of C. novyi-NT spores was given on Aug. 1, 2012 due tolack of biological activity seen with the prior IT doses. The onlyadverse event noted was Grade I hypertension after the 3rd IT dose.Hypertension was transient and self-limiting, resolving within 1 hour.Molly's tumor was surgically removed on Day 30 (Aug. 10, 2012) forhistologic analysis. The mass was confirmed to be a soft tissue sarcomawith areas of necrosis and inflammation. Bacteria were not present ongram stains, supporting lack of biological activity in this case.

Ricky (10-R01), a 13 year-old, male neutered, Golden retriever,presented with oral melanoma. His treatment history included surgeryprior to enrollment. He received 2 cycles of IT C. novyi-NT spores. C.novyi-NT IT treatments were administered on Aug. 2, 2012 and Aug. 9,2012. On Day 9 (Aug. 11, 2012), Ricky developed sudden onset of cervicalpain and rear leg neurological deficits 2 days after the 2nd treatmentcycle. Grade III anemia was also noted. An MRI was performed andrevealed probable cervical steatitis and cervical spinal cordcompression. Corticosteroids and gastrointestinal protectants wereadministered and Ricky recovered after 3 days. No changes in the oralmelanoma were noted and no additional C. novyi-NT treatments wereadministered. On Day 21 (Aug. 23, 2012), an MRI was performed and showedimprovement in the previously described steatitis; however, metastaticpulmonary nodules were noted on CT of the thorax. Excision of the oralmelanoma was performed. A human tyrosinase melanoma vaccine was startedon Aug. 30, 2012. On Day 42 (Sep. 13, 2012), Ricky presented withrecurrent cervical pain and forelimb pain (2 weeks after discontinuationof corticosteroids) and 2 weeks after receiving the melanoma vaccine.Medical management with pain medication did not result in improvementafter 4 days so corticosteroids were restarted. On Day 46, Grade IIIanemia and elevated BUN were noted. A presumptive gastrointestinal bleedwas treated with gastrointestinal protectants. On Day 60, Rickycollapsed and developed hematemesis. Humane euthanasia was performed. Anecropsy revealed disseminated metastatic melanoma includingsubmandibular lymph node, mediastinal lymph node, mesenteric lymph node,kidney, and perispinal fat in the region of the cervical spine. Noevidence of gastric or intestinal ulceration was found. The presumedcause for the two episodes of spinal pain is metastatic melanoma. Therelationship to C. novyi-NT is uncertain.

Finnegan (04-R02), an 11 year-old, entire male, Golden Retriever,presented with a soft tissue sarcoma (hemangiopericytoma) on the rightlateral metacarpus. His treatment history included surgery prior toenrollment. He received 4 cycles of IT C. novyi-NT spores. Adverseevents were mild and well tolerated. Complete ablation of the tumoroccurred after 4 cycles of treatment, leaving a margin of normal tissueabout the site of the tumor. Finnegan received his 1st, 2nd, 3rd and 4thtreatment cycles on Aug. 3, 2012, Aug. 10, 2012, Aug. 17, 2012 and Aug.24, 2012, respectively. Administration of C. novyi-NT was associatedwith only Grade I adverse events reported after the 1st, 2nd and 3rdcycles. Grade I and II adverse events were noted 48 hours after the 4thdose. Tumor infection was noted and consisted of fever, leukocytosis,neutrophilia and tumor-associated pain and abscess formation. Infectionprogressed to abscess formation and ablation of the entire tumor withminimal debridement occurring 96 hours after the 4th dose. Tumormeasurements at this visit were recorded in the morning prior tocomplete ablation of gross tumor later that day. Amputation of the limbwas pursued instead of open-wound management on Day 25 (Aug. 28, 2012)and antibiotics were given. Finnegan recovered uneventfully from surgeryand remains grossly tumor free 94 days (Nov. 5, 2012) after his firsttreatment.

Drake (04-R01, FIG. 10A), a 7 year-old, neutered male, Golden Retriever,presented with a soft tissue sarcoma (fibrosarcoma) in the right midmaxillary region. He had no treatment history prior to enrollment. Hereceived 4 cycles of IT C. novyi-NT spores. Adverse events were mild andwell tolerated. Complete ablation of the tumor occurred after 4 cycles,leaving a margin of normal tissue about the site of the tumor. Drakereceived his 1st, 2nd, 3rd, and 4th treatments on Aug. 13, 2012, Aug.20, 2012, Aug. 27, 2012, and Sep. 4, 2012, respectively. The intervalsbetween 1st, 2nd, and 3rd doses were 7 days; while the interval between3rd and 4th doses was 8 days in observance of a national holiday.Administration of C. novyi-NT was associated with mild adverse events,including Grade I lethargy and inappetence and Grade II vomiting andhematochezia reported 24-48 hours after the 1st cycle. These AEs weretreated successfully with an anti-emetic and antibiotic. AEs were notedwithin 24 hours of the 4^(th) dose, including Grade I tumor pain andswelling. Further evidence of tumor infection and abscess formation wasnot observed. Ablation of the tumor was evident on day 60 (Oct. 12,2012) and the tumor was not measurable (see FIG. 10B for a time courseof tumor measurements over the course of the study). The region was firmand remained slightly swollen and a CT scan was performed. Drake remainsfree of tumor on day 86 (Nov. 7, 2012) after 1st dose.

Baxter (04-R03, FIGS. 8A-F), a 9 year-old, neutered male, Boxer,presented with a grade II soft tissue sarcoma on the left medialantebrachium. He had no treatment history prior to enrollment. Hereceived three cycles of IT C. novyi-NT spores. Adverse events were mildand well tolerated. Complete ablation of the tumor occurred after threeinjections, leaving a margin of normal tissue about the site of thetumor. Baxter received his 1st, 2nd and 3rd doses of C. novyi-NT sporeson Aug. 17, 2012, Aug. 24, 2012, and Aug. 31, 2012, respectively.Administration of C. novyi-NT was well tolerated, with no study agentrelated toxicity reported after the 1st or 2nd dose. Study-relatedadverse events were noted 24 hours after the 3rd dose. These adverseevents were associated with tumor infection and consisted of fever,anorexia, lethargy and tumor-associated pain, swelling and bleeding.Adverse events were mild (Grade II or lower) and were managed withsupportive care and analgesics. C. novyi-NT related tumor infectionprogressed to involve the entire tumor and abscess formation. Surgicaldebridement of the tumor on Sep. 2, 2012 resulted in rapid resolution ofAEs. Wound healing was without complication and complete by Oct. 16,2012. Baxter remains grossly tumor free at 94 days (Nov. 19, 2012) afterhis first treatment (see FIG. 11 for a time course of tumor measurementsover the course of the study).

Harley (26-R01), a 7 year-old, neutered male, Labrador Retriever,presented with a grade II soft tissue sarcoma (hemangiopericytoma) onthe right paw. He had no treatment history prior to enrollment. Hereceived 4 cycles of IT C. novyi-NT spores. The 1st, 2nd, 3rd and 4thdoses were given on Aug. 20, 2012, Aug. 27, 2012, Sep. 4, 2012 and Sep.10, 2012. The interval between doses was 6-8 days. A baseline elevationof temperature was noted at the time of the 1st and 2nd doses. ITtreatment of C. novyi-NT spores was well tolerated with no adverseevents reported. There was no response to therapy.

Ursula (04-R-04), an 11 year old, female spayed, Saint Bernard mix,presented with chondroblastic osteosarcoma of the right forelimb. Hertreatment history included surgery prior to enrollment. She received asingle IT dose of C. novyi-NT spores. No metastatic disease was presentat enrollment. Following the first treatment on Aug. 31, 2012, tumorabscess formation and peritumoral inflammation was evident within thefirst 24 hours and medically managed with pain medication, warmcompresses and intravenous crystalloids. After no improvement, thetumor/abscess was lanced on Day 2 (Sep. 2, 2012). Moderateserosanguineous fluid was present. An anaerobic culture isolated C.novyi. Antibiotics were administered starting on Day 4 (Sep. 4, 2012).The incision was managed as an open wound until Day 20 (Sep. 20, 2012)when amputation was pursued for progressive disease. Histopathologyrevealed severe necrosis and hemorrhage along with persistingchondroblastic osteosarcoma. Following amputation, an incision siteinfection was noted. Cultures did not reveal C. novyi. No adjuvanttherapy was pursued following amputation. On Day 81 (Nov. 21, 2012),Ursula presented for rectal prolapse and was found to have rectalpolyps. Thoracic radiographs performed at the time of this evaluationrevealed pulmonary metastasis.

Gabriel (16-R02), a 9 year-old, neutered male, Labrador Retriever,presented with a grade I soft tissue sarcoma on the left lateral thigh.His treatment history included surgery prior to enrollment. He received4 cycles of IT C. novyi-NT spores. IT administration of C. novyi-NT wasgenerally well tolerated with a 1 week delay between the 1st and 2nddoses due to Grade II diarrhea that responded to medical management.Gabriel received his 1st, 2nd, 3rd and 4th doses on Sep. 12, 2012, Sep.26, 2012, Oct. 3, 2012 and Oct. 10, 2012 respectively. Toxicity was mildand consisted mainly of diarrhea and constitutive symptoms. Grade IIdiarrhea was noted after each dose and responded well to medicalmanagement. After the 1st dose, a 1-week dose delay was implementedresulting in a 14 day interval between the 1st and 2nd doses. Dosedelays were not implemented for further doses for Grade II diarrhea.Additionally, Grade II tumor swelling was observed on Day 4 (Sep. 16,2012). Tumor size remained stable from DO (Sep. 12, 2012) to D63 (Nov.14, 2012), the most recent study visit.

Buddy (04-R05), a 13 year-old, neutered male, Shetland sheepdog,presented with soft tissue sarcoma (rhabdomyosarcoma) on the rightantebrachium. His treatment history included surgery, chemotherapy, anda previous C. novyi-NT clinical trial prior to enrollment. No metastaticdisease was noted at the time of study entry. He received 4 cycles of ITC. novyi-NT spores. Clinically significant adverse eventscontemporaneous with C. novyi-NT were isolated to a Grade IIIneutropenia and fever following the 3rd cycle of therapy. This eventresolved within 48 hours of medical management with intravenousantibiotics and fluid therapy. Buddy received his 1st, 2nd, 3rd and 4thtreatment cycles on Sep. 20, 2012, Sep. 27, 2012, Oct. 5, 2012, and Oct.12, 2012. Mild tumor inflammation (erythema, warmth, swelling) was notedassociated with 2 of the 4 cycles. A transient decrease in tumor sizewas noted at Day 4 (Sep. 24, 2012). A new non-target lesion was notednear the primary tumor site on Day 21 (Oct. 12, 2012). The primarytarget tumor was stable at Day 61.

Amber (16-R03), a 10 year-old, neutered female, Shepherd, presented witha grade I soft tissue sarcoma on the left paw, palmar and dorsalsurfaces. Her treatment history included surgery prior to enrollment.She received 4 cycles of IT C. novyi-NT spores. The 1st, 2nd, 3rd and4th doses were given on Sep. 26, 2012, Oct. 3, 2012, Oct. 15, 2012, andOct. 24, 2012. The interval between doses was 7-12 days. Amberexperienced Grade II tumor swelling and pain after her 1st and 2nddoses. Grade I inappetence was noted on Day 2 (Sep. 28, 2012). On Day 8(Oct. 4, 2012, 1 day after 2nd dose), a Grade I fever, Grade II tumorwarmth and Grade III lameness was noted. Her tumor was lanced andanalgesics were given. A Grade III diarrhea was noted on Day 11 (Oct. 7,2012) and managed medically. Due to the tumor associated adverse eventsand diarrhea, the 3rd dose was delayed until Day 19 (Oct. 15, 2012).Grade II tumor swelling was again observed on Day 19, after the 3rd doseof C. novyi-NT and this was managed with analgesics. No adverse eventswere noted after the 4th dose.

Six (11-R04), a 9 year-old, neutered male, Husky, presented with a gradeI soft tissue sarcoma on the right paw. She had no treatment historyprior to enrollment. She received 4 cycles of IT C. novyi-NT spores. Sixreceived the 1st, 2nd, 3rd and 4th doses on Oct. 1, 2012, Oct. 8, 2012,Oct. 15, 2012, and Oct. 22, 2012, respectively. Administration of C.novyi-NT spores was well tolerated with only mild adverse eventsobserved. After the 1st dose, Grade I hypertension and fever were noted.Fever and hypertension were self-limiting and resolved within 1 and 2hours of dosing respectively. On Day 4 (Oct. 5, 2012), the tumor wassubjectively softer and a small area of ulceration (Grade I) wasobserved at the site of a previous biopsy. Ulceration continued to Day31 (Nov. 1, 2012), the most current study visit. This ulceration may beassociated with either the study agent or a complication of the biopsyrequired for study enrollment.

Belle (04-R06), an 11 year-old, female spayed, Labrador retriever,presented with a mast cell tumor (originally aspirated as a soft tissuesarcoma) on the right rear digit 3 with metastasis to the popliteallymph node. She had no treatment history prior to enrollment. Shereceived 4 cycles of IT C. novyi-NT spores. Adverse events were mild andlimited to Grade I fever and Grade I tumor inflammation. Belle receivedthe 1st, 2nd, 3rd and 4th treatment cycles on Oct. 19, 2012, Oct. 26,2012, Nov. 2, 2012, and Nov. 9, 2012. Grade I fever contemporaneous withC. novyi-NT treatment and tumor inflammation. Fever and inflammationwere self-resolving without the need for medical management other thanprotocol required subcutaneous fluids administered on scheduled studyvisits. Ulceration of the tumor was noted on Day 21 (Nov. 9, 2012).Photographs of the tumor sent to the investigator by the dog ownershowed resolution of the ulceration and marked regression in the mass.An unscheduled visit was performed on Day 46 (Dec. 4, 2012) to capturetumor response assessment. Complete regression of the tumor was noted.

Frida (11-R01), a 7 year-old, female spayed, German shepherd mix,presented with a soft tissue sarcoma (hemangiopericytoma) on the rightrear paw with possible lymph node metastasis (based on CT). Hertreatment history included surgery prior to enrollment. She traveledwith her owner from Mexico to participate in this clinical trial. Shereceived 3 cycles of IT C. novyi-NT spores. Adverse events were limitedto a waxing and waning fever for 48 hours, which resolved withintravenous fluids and NSAIDs. Frida received the 1st, 2nd, and 3rdcycles of therapy on Nov. 6, 2012, Nov. 14, 2012, and Nov. 21, 2012. Theonly significant adverse events included Grade I fever requiringhospitalization and fluids starting on Day 4 (Nov. 10, 2012) andprogressing to Grade II fever on Day 5 (Nov. 11, 2012). The feverresolved after 48 hours. A Grade I fever was also noted after the 3rdcycle of therapy on Day 18 (Nov. 24, 2012). Tumor progression promptedamputation on Day 21 (Nov. 27, 12).

Mhija (01-R02), a 7 year-old, neutered male, Border Collie, presentedwith soft tissue sarcoma (peripheral nerve sheath tumor) on the leftthoracic flank. She had no treatment history prior to enrollment. Shehas received 3 cycles of IT C. novyi-NT spores. Adverse events were mildand well tolerated. Tumor inflammation, heat and serosanguineous tomucopurulent discharge are probably related to C. novyi-NT activity. A4th cycle of C. novyi-NT spores is planned. Mhija received the 1st, 2ndand 3rd doses on Nov. 12, 2012, Nov. 20, 2012, and Nov. 27, 2012,respectively. The interval between 1st and 2nd doses was 8 days; whilethe interval between 2nd and 3rd doses was 7 days. Administration of C.novyi-NT was associated with mild, Grade I-II toxicity. Grade I nauseaand regurgitation was noted after the 1st dose, with Grade I inappetenceand lethargy noted after the 3rd dose. Toxicities resolved shortly withmedical management. Most toxicities were localized to the tumor site,Grade I or II in severity (heat, inflammation, pruritis, serosanguineousto mucopurulent discharge and erythema) and occurring within 2 days ofan administration of C. novyi-NT. Additionally, Grade I-II ventral edemawas observed 2 days after the 1st and 3rd doses.

Tank (10-R02), a 10 year-old, male neutered, mixbreed, presented withsoft tissue sarcoma (hemangiopericytoma) on the right flank. Histreatment history included surgery prior to enrollment. He received 1cycle of IT C. novyi-NT spores on Nov. 12, 2012. Grade I fever,decreased appetite, Grade II edema surrounding the tumor, and Grade IIItumor abscess were noted on Day 4 (Nov. 16, 2012) following treatment.Medical management including pain medication, IV fluids, andbroad-spectrum antibiotics were used to manage the abscess. Tumorinflammation and surrounding edema resolved on Day 11 (Nov. 23, 2012).Tank received a 2nd treatment cycle on Dec. 3, 2012. The intervalbetween cycles was 21 days. The 2nd dose was delayed due to theantibiotics washout period.

Time courses of tumor measurements from eight of the dogs are shown inFIG. 12A. FIG. 12B shows three time courses that were shortened due toamputation or data cut-off.

In summary, C. novyi-NT administered by IT injection at a dose of 1×10⁸spores per cycle with up to 4 cycles of treatment exhibits meaningfulbiological and anti-tumor activities and appears to be well-tolerated incompanion dogs with naturally occurring solid tumors. Tumor responsesare rapid, with significant tumor necrosis and notable diseaseregression occurring within days of C. novyi-NT administration. Mostadverse events are limited to Grade 1 and Grade 2, and are consistentwith the mechanism-based tumor inflammatory reactions expected from theC. novyi-NT therapeutic. Several cases are currently under long-termfollow-up for assessment of progression and survival.

Example 8 Intratumoral (IT) Administration of C. novyi-NT—Study 2Methods

A study characterizing dose and volume of C. novyi-NT administration byIT injection for the treatment of dogs with solid tumors (excludingosteosarcoma or mast cell tumor) is being performed.

Dogs with solid tumors (except osteosarcoma or mast cell tumor) of anyweight, breed, sex, or age were screened for enrollment. Inclusioncriteria was similar to that presented in Example 6, with the exceptionthat each dog had a cytologic or histologic diagnosis of any cancerexcluding osteosarcoma or mast cell tumor, and that each dog had atleast 1 measurable tumor lesion with a longest diameter 1 cm.

During the initial screening visit each dog was assigned a unique studydog identification number consisting of a 5-digit numeric code (whichmay not be sequentially in order of the screening dog number). The first2 digits indicated the study site (01 to 99), the middle digit indicatedthe study ‘5’, and the last 2 digits described the study dog numberwithin a study site (01 to 99). For example the 11th dog enrolled atSite 9 was assigned study dog number 09-511. Study dog numbers wereassigned chronologically in the order that dogs were enrolled at a givenstudy site. A dog was considered enrolled in the study when it satisfiedthe inclusion and exclusion criteria.

Gross pathology, histopathology, and necropsy were performed asdescribed in Example 6.

C. novyi-NT spores were prepared as set forth above prior to shipment ata concentration of 1×10⁸ spores/mL and suspended in sterile saline in 2mL cryovials. Each cycle of C. novyi treatment was composed of up to 5injections of 1 mL spore suspension (1×10⁸ spores) for each injectioninto a single target lesion. The spore suspension containing 1×10⁸spores was packed in individual cryovials for each 1 mL injection, andthe vial, syringe, and needle were discarded after each injection.

The scheme for injection is shown in FIG. 13. Five 1 mL injection sites(as represented by squares) were distributed within the tumor: center,and four (4) evenly allocated injection sites within the tumor. The sitefor each 1 mL injection further consisted of 5 redirection sites (asrepresented by circles in FIG. 13). Each redirection site received 200μL of spore suspension. The needle was first directed within the centerof the injection site, and then evenly redirected to the four corners ofthe injection site without withdrawing the needle. Upon the completionof the first 1 mL injection, the needle was withdrawn and the syringewas discarded. The depth of each injection should be adequatelydistributed such that the best distribution is achieved. The recommendedsize of syringe was 1 mL for each injection, the recommended needle wasbetween 22-gauge and 25-gauge. Adequate length of needle should beselected based on the depth of the tumor lesion.

All dogs were hospitalized from DO to D2, and then at the Investigator'sdiscretion for 24 to 48 hours after each subsequent treatment forclinical observation. Fluids were administered to all study dogs duringhospitalization following C. novyi-NT treatment. On dosing days all dogswere administered IV crystalloids at 4 ml/hg/h for 2 hourspost-treatment with C. novyi-NT.

Study visits and events are summarized in Table 18, as an example of an8-cycle treatment regimen. The dosing interval was suggested to beweekly if the intent was to treat the dog with multiple cycles oftherapy.

TABLE 18 Summary of study visits and events Screen Cycle D-14 to Cycle1* 2-8† D 0 D 0 D_(—) D 70 ± 7 days D 90 ± 7 days Informed consent XDemographics X Weight and vitals X X X X X Physical examination X X X XX Lab samples X X (X) (X) X Research blood X X X X X samples Researchtumor sample X Diagnostic imaging X   X*** Performance score XInclusion/exclusion X Enrollment X Tumor measurement X X X X X C.novyi-NT*  x*  x† Crystalloids**  x**  x** Study completion  X†† *Ownerswill leave their dog in clinic from the D 0 until D 2, and IVcrystalloids will be administered to all dogs in hospital. Forsubsequent cycles, Investigators will fill in the D according to thenumber of days on study, relative to D 0. **Dogs will be administered IVcrystalloids. ***Thoracic radiographs only. †Dogs may not receive 8cycles. For this study, the decision to continue subsequent cycle ofdosing will be made on a case by case basis via consultation among theMedical Director, Investigator and Sponsor. ††Following study completionand if systemic antibiotics were required to manage adverse events, itis recommended to administer doxycycline 5-10 mg/kg PO BID to dogs for 3months.

Example 9 Intratumoral (IT) Administration of C. novyi-NT—Study 2Interim Results

As of Dec. 2, 2012, two companion dogs have been treated in the study.Both animals received a dose level of 5×10⁸ spores administered at 5unique IT injection sites per treatment cycle.

The first dog, Buddy (04-503), a 9 year-old, male neutered, Belgianmalinois, presented with soft tissue sarcoma on the left carpus with aLD measurement of 69 mm at baseline (4.4×3.3×0.7 cm by CT). Histreatment history included surgery prior to enrollment. He received 2cycles of IT C. novyi-NT spores. Adverse events were mild and limited toGrade I fever and Grade I tumor inflammation. Buddy received the 1st and2nd treatment cycles on Nov. 21, 2012 and Nov. 28, 2012. Grade I feverand tumor redness, swelling and increased pain were noted within 6 hoursof the first injection. The fever resolved within 6 hours followingtreatment with the NSAID carprofen. Mild tumor ulceration was noted onDay 2 (Nov. 23, 2012) following treatment. At Day 7 (Nov. 28, 2012), aslight decrease in the size of the mass was noted (−12.0%). Each cycleof treatment was well tolerated with no adverse events greater thanGrade I.

The second dog, Guinness (04-502), a 9 year-old, male neutered, Wheatonterrier, presented with squamous cell carcinoma on the left shoulderwith a LD measurement of 122 mm at baseline (9.1×9.3×14.5 cm by CT), alow-grade hemangiosarcoma on the rear leg, and evidence of pulmonarymetastasis (based on CT). His treatment history included surgery priorto enrollment. Preexisting mitral valve disease was evident based onechocardiography performed prior to enrollment. He received a singledose of IT C. novyi-NT spores on Nov. 28, 2012. Grade III fever wasnoted within 6 hours of treatment and medically managed with IV fluids.On Day 1 (Nov. 29, 2012), abscess of the mass, purulent discharge, andneutrophilia were appreciated. IV fluids were continued and painmedications (including NSAIDs) were started. On Day 2 (Nov. 30, 2012),progressive tumor swelling and evidence of sepsis (fever, neutropenia,hypoglycemia, hypoalbuminemia) prompted lancing of the tumor andirrigation. Broad-spectrum antibiotics, hetastarch and human albuminwere administered. On Day 3 (Dec. 1, 2012), progressive decline instatus was noted resulting in respiratory distress. Euthanasia solutionwas administered. A necropsy was performed. Gross clinically significantfindings included vegetative endocarditis, suppurative lung nodules, andwhole-body subcutaneous hemorrhage and edema. Postmortem aerobiccultures from various tissues and organs (lung, liver, heart, kidney,spleen, GI, stomach) revealed polymicrobial bacterial growth(Staphylococcus aureus, Pseudomonas aeruginosa, E. coli, Streptococcusspecies); anaerobic cultures from all organs and tissues were negativefor C. novyi-NT growth except in the tumor tissue and urinary bladder.Histopathology of affected tissues are pending. Septic toxemia shock isconsidered the most likely cause of death and relationship to C.novyi-NT therapy is unknown at this time.

Example 10 Intratumoral (IT) Administration of C. novyi-NT inHumans—Methods

Phase I Human Clinical Trial of IT Injected C. novyi-NT Spores

An open-label, non-randomized, multi-center phase I safety study of asingle IT injection of C. novyi-NT spores is currently ongoing inpatients with treatment-refractory solid tumors. The clinical studyprotocol was reviewed and approved by the Institutional Review Board(IRB) of each participating institution, and all regulatory steps wereperformed under the guidance of the Food and Drug Administration (FDA)(number NCT01924689). All patients were required to sign a writtenInformed Consent Form (ICF) before inclusion in the study.

The primary objectives of this phase I study were to determine thesafety profile, dose limiting toxicities (DLT), and maximum tolerateddose (MTD) of IT injected C. novyi-NT. In addition, the anti-tumoractivity of the therapeutic was explored.

Preparation and IT Injection of C. novyi-NT Spores in Phase I Study

The clinical supply of C. novyi-NT spores was packaged in a single-use 2mL sterile and pyrogen-free, Type I borosilicate glass vial with arubber stopper and aluminum seal with a tamper resistant cap at aconcentration of 8.52×10⁸ spores/mL suspended in sterile phosphatebuffered saline (PBS) with a 1.0 mL fill volume. The vials were storedbetween 2-8° C. in controlled temperature environment under constanttemperature monitoring. The GMP product was manufactured and formulatedby Omnia Biologics, Inc. (Rockville, Md.).

After a patient was enrolled in the trial, one vial was shipped to thestudy site. Further preparation of C. novyi-NT was required and occurredon the same day of the IT injection. Dilution of the concentrated sporesuspension was performed in a designated biological safety cabinet usingsterile saline (0.9%) infusion bags of appropriate size to achieve therequired dose based on the assigned cohort. The injection volume (3 mL)was then withdrawn from the saline bag and injected under radiographicguidance. C. novyi-NT spores were injected with an 18-gauge multi-prongneedle (Quadra-Fuse®, Rex-Medical, Conshohocken, Pa.).

Design and Conduct of Human Clinical Trial

The study was conducted with a standard 3+3 dose-escalation design.Patients must have been diagnosed as having an advanced solid tumormalignancy with a target tumor that was measureable, palpable or clearlyidentifiable under ultrasound or radiographic guidance and amenable topercutaneous injection of C. novyi-NT spores. The targeted lesion musthave a longest diameter ≥1 cm and be measurable as defined by RECIST 1.1criteria. The main eligibility criteria included history of a treatmentrefractory malignancy; age of at least 18 years; Eastern CooperativeOncology Group (ECOG) performance status ≤2; able to stay within 45minutes driving time of an emergency room and having a caregiver for 28days after IT injection. The main exclusion criteria were pregnancy;primary brain malignancy or brain metastases; clinically significantascites or clinical evidence or history of portosystemic hypertension orcirrhosis; Glasgow Coma Score (GCS)<15; serum creatinine level >1.5× theupper limit of normal (ULN), chronic renal failure requiringhemodialysis or peritoneal dialysis; oxygen saturation (SpO₂)<95% (roomair); mean arterial blood pressure (BP) <70 mmHg; platelet count<100,000/mm³; hemoglobin <9.0 g/dL; absolute neutrophil count(ANC)<1,000/mm³; clinically significant pleural effusion, pericardialeffusion, circumferential pericardial effusion, or any effusion greaterthan 1.0 cm at any location around the heart; need to ongoing treatmentwith an immunosuppressive agent; history of solid organ transplantation;systemic or localized infection.

Eligible patients were admitted and enrolled into a dose cohort. Underthe protocol, patients remain hospitalized after spore administrationand observed for 8 days, and patients return to the clinical site forroutinely scheduled follow-up visits for 12 months, during which timeassessments of safety and efficacy were performed.

Clinical response and progression were evaluated using the RECISTversion 1.1. Objective responses were measured by serial CT or MRI scansof the injected tumor, as well as distant metastases (up to 5 targetlesions). Safety monitoring for infectious complications or othertreatment-emergent adverse events were continuously conducted for 12months.

Example 11 Intratumoral (IT) Administration of C. novyi-NT inHumans—Results

C. novyi-NT Causes Rapid Local Tumor Destruction in the First HumanPatient

The promising outcomes and favorable risk/benefit profiles of biosurgeryin the comparative canine trial, in conjunction with the resultsobserved in rats, provided a rationale for attempting biosurgery inhumans. Accordingly, a Phase I investigational study in human patientswith solid tumors that were either refractory to standard therapy orwithout an available standard therapy was initiated (NCT01924689). Thefirst patient enrolled in this trial is reported herein: a 53-year-oldfemale diagnosed with a retroperitoneal leiomyosarcoma in August 2006.The patient underwent several surgical resections and received multiplechemotherapy and radiotherapy treatments, including a right radicalnephrectomy and radiation therapy in March 2007, chemotherapy withgemcitabine, taxol, adriamycin, and ifosfamide, resection of livermetastasis in November 2008, multiple wedge resections of right-sidedpulmonary metastases in December 2009, trabectedin treatment from March2010 to April 2011, multiple wedge resection of left-sided pulmonarymetastases in December 2010, pazopanib treatment in April 2011, leftlower lobectomy in October 2011, HAI abraxane, gemcitabine, and avastinfrom February 2012 to January 2013, everolimus and pazopanib fromFebruary 2013 to July 2013, and bland arterial hepatic embolization inAugust 2013 and September 2013. However, the patient progressed, withmetastatic disease present in her liver, lungs, peritoneum, and softtissue in the right shoulder and adjacent right humerus.

Biosurgery was performed with the planned starting dose of 1×10⁴ C.novyi-NT spores injected into her metastatic right shoulder tumor withan 18-gauge multi-prong needle (day 0, Nov. 19, 2013).

CT-Guided Intratumoral Injection Using a Three-Pronged Needle

The subject was placed under moderate sedation with fentanyl and versedfor 35 minutes. An 18-gauge Quadra-Fuse device (Rex Medical) (FIG. 16A)was employed for injection under CT guidance by inserting the 3-prongedneedle (27 g) in the target injection area (FIGS. 16B and 16C). Threetines (each having 2 through holes, for 4 fluid exits) (FIG. 16D) weredeployed at 4, 3, and 2 cm at which location (FIG. 16E), a 1 ml aliquotof C. novyi-NT spore solution was injected during the staged retractionprocess. The device was removed after the deployed tines were fullyretracted into the needle cannula and manual compression was utilized toachieve hemostasis.

On day 1, the patient experienced mild right shoulder pain extending tothe scapula, which responded to tramadol and acetaminophen. On day 2,her pain required IV patient controlled analgesia with hydromorphone,her leukocyte count increased to 18,300 per μL, and she developed feverwith a maximum temperature of 39.2° C. On day 3, the pain in thepatient's right shoulder and scapula was difficult to control. Hermaximum temperature was 37.8° C. The CT scan of the right upperextremity demonstrated extensive tumor destruction with gas in the softtissue and bony component of the tumor (FIG. 14A). Necrosis of herhumerus was discussed. A CT-guided aspirate of her tumor revealed C.novyi-NT growth under anaerobic culture conditions. The patient was thenstarted on antibiotics and defervesced shortly after. On day 4, a MRI ofthe right upper extremity demonstrated markedly diminished enhancementconfined to the tumor mass compared to baseline (FIGS. 14B and 14C).Biopsies from the tumor showed many gram-positive bacteria and anabsence of viable tumor cells. At the time of the biopsies, apercutaneous drain was placed within the tumor abscess to drain fluidand debris. The patient remained afebrile and her leukocyte countgradually normalized. She continued on antibiotics and was kept in thehospital for IV analgesia until day 20 when she was transitioned to oralanalgesics. She was discharged on orally administered metronidazole anddoxycycline per protocol. On day 29, a follow-up MRI demonstrated anongoing reduction in tumor enhancement (FIG. 14D). On day 55 the patientpresented with localized pain as a result of a patient-effort inducedpathologic fracture of the right proximal humerus. Subsequent partialresection of the humerus, debridement, and internal fixation with anintramedullary nail and cement spacer resulted in significantimprovement in pain and an increase in range of motion. Intraoperativecultures revealed C. novyi-NT growth under anaerobic culture conditions.Histopathology demonstrated extensive tumor necrosis with small foci ofresidual tumor cells. (FIGS. 15A-D). The patient continues to bemonitored and has a performance status of 1 on the Eastern CooperativeOncology Group scale (ECOG) with no clinical signs of infection.

DOCUMENTS

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All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

1-62. (canceled)
 63. A method for debulking or abalating a solid tumorpresent in a human comprising administering intratumorally to the humana unit dose of C. novyi colony forming units (CFUs) comprising1×10³-1×10⁶ CFUs suspended in a pharmaceutically acceptable carrier orsolution, wherein the C. novyi is effective to debulk or ablate thesolid tumor without administration of additional anti-cancer agents. 64.The method according to claim 63, wherein the solid tumor is selectedfrom the group consisting of soft tissue sarcoma, hepatocellularcarcinoma, breast cancer, pancreatic cancer, and melanoma.
 65. Themethod according to claim 63, wherein the solid tumor is leiomyosarcoma.66. The method according to claim 65, wherein the solid tumor isretroperitoneal leiomyosarcoma.
 67. The method according to claim 63,wherein the unit dose comprises about 1×10⁴-1×10⁶ C. novyi CFUs.
 68. Themethod according to claim 63, wherein the C. novyi CFUs are selectedfrom the group consisting of vegetative and spore forms.
 69. The methodaccording to claim 63, wherein the C. novyi is C. novyi NT.
 70. Themethod according to claim 69, wherein the unit dose comprises1×10⁴-1×10⁶ C. novyi NT spores.
 71. The method according to claim 70,wherein the unit dose comprises 1×10⁵-1×10⁶ C. novyi NT spores.
 72. Themethod according to claim 63, wherein the administering step comprisesinjecting the unit dose at a single location into the tumor.
 73. Themethod according to claim 63, wherein the administering step comprisesinjecting the unit dose at multiple unique locations into the tumor. 74.The method according to claim 63, wherein the administering stepcomprises injecting the unit dose at 1-5 unique locations into thetumor.
 75. The method according to claim 63, wherein the administeringstep comprises injecting the unit dose at 5 or more unique locationsinto the tumor.
 76. The method according to claim 63 further comprisingadministering a plurality of treatment cycles to the human, eachtreatment cycle comprising injecting one unit dose of the C. novyi CFUsinto the solid tumor.
 77. The method according to claim 76, wherein 2-10treatment cycles are administered.
 78. The method according to claim 76,wherein 2-4 treatment cycles are administered.
 79. The method accordingto claim 76, wherein an interval between each treatment cycle is about5-100 days.
 80. The method according to claim 76, wherein an intervalbetween each treatment cycle is about 7 days.
 81. The method accordingto claim 69 further comprising administering IV fluids to the humanbefore, during, and/or after each administration of the C. novyi NTspores.
 82. The method according to claim 69 further comprisingadministering a plurality of treatment cycles to the human, eachtreatment cycle comprising injecting one unit dose of the C. novyi NTspores into the solid tumor.
 83. The method according to claim 82,wherein 2-4 treatment cycles are administered.
 84. The method accordingto claim 63 further comprising administering IV fluids to the humanbefore, during, and/or after each administration of the C. novyi. 85.The method according to claim 63 further comprising providing the humanwith a first course of antibiotics for a period of time and at a dosagethat is effective to treat or alleviate an adverse side effect selectedfrom the group consisting of infections, vomiting, hematochezia, fever,and combinations thereof caused by the C. novyi.
 86. The methodaccording to claim 85, wherein the antibiotics are administered for twoweeks post C. novyi administration.
 87. The method according to claim85, wherein the antibiotics are selected from the group consisting ofamoxicillin, clavulanate, metronidazole, and combinations thereof. 88.The method according to claim 85 further comprising providing the humanwith a second course of antibiotics for a period of time and at a dosagethat is effective to treat or alleviate an adverse side effect selectedfrom the group consisting of infections, vomiting, hematochezia, fever,and combinations thereof caused by the C. novyi.
 89. The methodaccording to claim 88, wherein the second course of antibiotics isinitiated after completion of the first course of antibiotics and iscarried out for 1-6 months.
 90. The method according to claim 88,wherein the second course of antibiotics is initiated after completionof the first course of antibiotics and is carried out for 3 months. 91.The method according to claim 88, wherein the antibiotic used in thesecond course is doxycycline.
 92. The method according to claim 63,further comprising administering to the human an anti-cancer agentselected from the group consisting of chemotherapy, radiation therapy,immunotherapy, and combinations thereof, after the C. novyi have actedto debulk or ablate the solid tumor.
 93. The method according to claim92, wherein the immunotherapy comprises administering to the human animmune checkpoint inhibitor.
 94. The method according to claim 63,wherein the solid tumor is resistant to a therapy selected from thegroup consisting of chemotherapy, radiation therapy, immunotherapy, andcombinations thereof.
 95. The method according to claim 92, wherein thechemotherapy comprises administering to the human an agent selected fromthe group consisting of an anti-metabolite, a microtubule inhibitor, aDNA damaging agent, an antibiotic, an anti-angiogenesis agent, avascular disrupting agent, a molecularly targeted agent, andcombinations thereof.
 96. The method according to claim 92, wherein thechemotherapy comprises administering to the human an agent selected fromthe group consisting of gemcitabine, taxol, adriamycin, ifosfamide,trabectedin, pazopanib, abraxane, avastin, everolimus, and combinationsthereof.
 97. The method according to claim 63, wherein the solid tumoris refractory to standard therapy or the solid tumor is without anavailable standard therapy.
 98. The method according to claim 63,wherein the unit dose of C. novyi induces a potent localizedinflammatory response and an adaptive immune response in the human. 99.A method for microscopically precise excision of tumor cells in a humancomprising administering intratumorally to the human a unit dose of C.novyi NT colony forming units (CFUs) comprising 1×10³-1×10⁶ CFUssuspended in a pharmaceutically acceptable carrier or solution, whereinthe C. novyi is effective for microscopically precise excision of thetumor cells without administration of additional anti-cancer agents.100. A method for debulking or ablating a solid tumor that hasmetastasized to one or more sites in a human comprising administeringintratumorally to the human a unit dose of C. novyi NT colony formingunits (CFUs) comprising 1×10³-1×10⁶ CFUs suspended in a pharmaceuticallyacceptable carrier or solution, wherein the C. novyi is effective todebulk or ablate the solid tumor without administration of additionalanti-cancer agents.
 101. The method according to claim 100, wherein atleast one site is distal to the original solid tumor.
 102. A method fordebulking a solid tumor present in a human comprising administeringintratumorally to the human a unit dose of C. novyi CFUs comprising1×10³-1×10⁶ CFUs suspended in a pharmaceutically acceptable carrier orsolution, wherein the C. novyi is effective to debulk the solid tumorwithout administration of additional anti-cancer agents.
 103. The methodaccording to claim 102, wherein the solid tumor is selected from thegroup consisting of soft tissue sarcoma, hepatocellular carcinoma,breast cancer, pancreatic cancer, and melanoma.
 104. A method fordebulking a solid tumor present in a human comprising administeringintratumorally to the human one to four cycles of a unit dose of C.novyi NT spores comprising 1×10³-1×10⁶ spores per cycle, each unit doseof C. novyi NT being suspended in a pharmaceutically acceptable carrieror solution, wherein the C. novyi is effective to debulk the solid tumorwithout administration of additional anti-cancer agents.
 105. A methodfor debulking or ablating a solid tumor present in a human comprisingadministering intratumorally to the human one to four cycles of a unitdose of C. novyi NT spores comprising 1×10³-1×10⁶ spores per cycle, eachunit dose of C. novyi NT spores being suspended in a pharmaceuticallyacceptable carrier or solution, wherein the C. novyi is effective todebulk or ablate the solid tumor without administration of additionalanti-cancer agents.
 106. A method for ablating a solid tumor present ina human comprising administering intratumorally to the human a unit doseof C. novyi CFUs comprising 1×10³-1×10⁶ CFUs suspended in apharmaceutically acceptable carrier or solution, wherein the C. novyi iseffective to ablate the solid tumor without administration of additionalanti-cancer agents leaving a margin of normal tissue.
 107. The methodaccording to claim 106, wherein the tumor is a sarcoma.